UC Opp No. 2 CRISPR Patent

Filed on behalf of Senior Party
THE REGENTS OF THE UNIVERSITY OF CALIFORNIA,

UNIVERSITY OF VIENNA, AND EMMANUELLE CHARPENTIER
By:
Todd R. Walters, Esq.

Erin M. Dunston, Esq.
Travis W. Bliss, Ph.D., Esq.
BUCHANAN INGERSOLL & ROONEY PC
1737 King Street, Suite 500
Alexandria, Virginia 22314-2727
Telephone (703) 836-6620
Facsimile (703) 836-2021
todd.walters@bipc.com
erin.dunston@bipc.com
travis.bliss@bipc.com
By: Li-Hsien Rin-Laures, M.D., Esq.

Sandip H. Patel, Esq.
Greta Noland
MARSHALL GERSTEIN & BORUN LLP
6300 Willis Tower
233 South Wacker Drive
Chicago, Illinois 60606
Telephone (312) 474-6300
Facsimile (312) 474-0448
lrinlaures@marshallip.com
spatel@marshallip.com
gnoland@marshallip.com
UNITED STATES PATENT AND TRADEMARK OFFICE

____________________
BEFORE THE PATENT TRIAL AND APPEAL BOARD
____________________
THE BROAD INSTITUTE, INC., MASSACHUSETTS INSTITUTE OF
TECHNOLOGY, and PRESIDENT AND FELLOWS OF HARVARD COLLEGE
Patents 8,697,359; 8,771,945; 8,795,965; 8,865,406; 8,871,445; 8,889,356;
8,895,308; 8,906,616; 8,932,814; 8,945,839; 8,993,233; 8,999,641; and Application 14/704,551,
Junior Party,
v.
THE REGENTS OF THE UNIVERSITY OF CALIFORNIA, UNIVERSITY
OF VIENNA, AND EMMANUELLE CHARPENTIER,
Application 13/842,859,
Senior Party.
____________________
Patent Interference 106,048 (DK)
____________________
UC et al. OPPOSITION 2
(Opposing Broads Allegations of No Interference-In-Fact)
Interference No. 106,048

TABLE OF CONTENTS
Page
I.
INTRODUCTION ...............................................................................................................1
II.
THE EVIDENCE .................................................................................................................2
III.
STATEMENT OF MATERIAL FACTS.............................................................................2
IV.
ARGUMENT .......................................................................................................................2
A.
Broads Sole Argument is That There Would Have Been No Reasonable

Expectation of Success in Using the Type-II CRISPR-Cas System of UCs
Claims in a Eukaryotic Cell .....................................................................................2
B.
Broad Has the Burden of Proof ................................................................................3
C.
By Ignoring Prior Art, Broad Failed to Meet Its Burden of Proving that it
Would Not Have Been Obvious to Use UCs Type-II CRISPR-Cas System
in Eukaryotic Cells ...................................................................................................4
D.
Contemporaneous Evidence Demonstrates Motivation and Reasonable

Expectation of Success in Using the Type-II CRISPR-Cas System in
Eukaryotes................................................................................................................9
E.
1.
Multiple Researchers in the Art Had an Expectation of Success in

Adapting UCs System to Eukaryotic Cells Before Broad
Published Its Results ..................................................................................10
2.
Like All the Others, Broad Merely Made Obvious Modifications to

UCs Invention and Only After UCs Disclosure ......................................12
3.
The Contemporaneous Quotations Cited by Broad Further

Evidence Obviousness When Placed in Context .......................................14
Broad Fails to Address Extensive Evidence that the State of the Art
Rendered Use of the Type-II CRISPR-Cas System in Eukaryotic Cells
Obvious Once the System Was Described.............................................................16
1.
Broad Failed to Address the Fact that All of the Techniques Used
to Introduce the Type-II CRISPR-Cas System into Eukaryotic
Cells Were Routine ....................................................................................16
2.
Examples of Successful Use of Prokaryotic DNA-Targeting

Proteins in Eukaryotic Cells Provided a Reasonable Expectation
that the Prokaryotic DNA-Targeting Protein Cas9 Would Likewise
Function .....................................................................................................17
-i-
F.
V.
3.
Broads Arguments are Unsupported and Contradicted by

Evidence .....................................................................................................19
4.
Broads Arguments of Non-Specific Differences Between

Prokaryotes and Eukaryotes Do Not Dispel the Evidence of
Reasonable Expectation of Success ...........................................................20
5.
Broads Non-Analogous Examples Would Not Dispel the

Expectation of Successfully Using the Type-II CRISPR-Cas
System in Eukaryotic Cells ........................................................................24
Comments of UCs Inventors Do Not Contradict the Reasonable

Expectation of Success and Are Irrelevant ............................................................27
CONCLUSION ..................................................................................................................31
APPENDIX 1 - LIST OF EXHIBITS

APPENDIX 2 STATEMENT OF FACTS
- ii -

TABLE OF AUTHORITIES
Cases
Page(s)
Alarm.com v. Icontrol Networks, Inc.,

Interference No. 106,001, 2015 WL 1871503 (P.T.A.B. Mar. 31, 2015)........................3, 4
In re Droge,
695 F.3d 1334 (Fed. Cir. 2012)..........................................................................................28
KSR Intl Co. v. Teleflex Inc.,
127 S.Ct. 1727 (2007) ........................................................................................................12
In re Kubin,
561 F.3d 1351 (Fed. Cir. 2009)..........................................................................................30
In re Longi,
759 F.2d 887 (Fed. Cir. 1985)............................................................................................28
Par Pharm., Inc. v. TWi Pharm., Inc.,
773 F.3d 1186 (Fed. Cir. 2014)..........................................................................................28
Statutes
35 U.S.C. 102(e) ...........................................................................................................................6
Rules
37 C.F.R. 41.121(b) ......................................................................................................................3
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1
I.
INTRODUCTION
In Broad et al. Substantive Motion 2 (Broad Motion 2), Junior Party (Broad) moves
for judgment of no interference-in-fact between the Parties involved claims. The sole basis on
which Broad Motion 2 rests is an argument that persons of ordinary skill in the art would not
have had a reasonable expectation of success in using the Type-II CRISPR-Cas system disclosed
and claimed by Senior Party (UC) in eukaryotic cells.
Broad Motion 2 fails to meet its burden of proof by ignoring evidence to the contrary. A
scientific review and perspective on UCs first public disclosure of the Type-II CRISPR-Cas
system in Jinek et al., 337 SCIENCE 816-821 (2012) (Jinek 2012) (Ex. 1155), published
10
alongside it in the same issue of SCIENCE, both of which are prior art to Broad, credited Jinek et
11
al. with the realization and proof of concept that a highly specific, customizable RNA-directed
12
DNA nuclease could be useful to edit whole genomes by introducing breaks at unique sites in
13
any eukaryotic genome. Ex. 1471. Broad Motion 2 also ignores the fact that immediately
14
following Jinek 2012and before Broad had even filed its first provisional applicationanother
15
independent group filed a patent application citing Jinek 2012 as motivation and confirmed the
16
use of the Type-II CRISPR-Cas system in eukaryotic cells. Ex. 1545. In addition, manuscripts
17
demonstrating use of UCs Type-II CRISPR-Cas system in eukaryotic cells were submitted by
18
other groups before Broad filed its first provisional application or at about the same time.
19
Each of these independent groups was able to quickly adapt the Type-II CRISPR-Cas
20
system to eukaryotic cells because, like Broad, they used conventional techniques that were well-
21
known in the art and had been routinely used for many years to adapt other prokaryotic systems
22
to eukaryotic cells.
23
24
All of this objectively demonstrates that as soon as the Type-II CRISPR-Cas system
recited in UCs claims was disclosed, it was obvious to use it in eukaryotic cells.
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II.
2
3
THE EVIDENCE
A list of exhibits upon which this Opposition relies is set forth in Appendix 1.
III.
STATEMENT OF MATERIAL FACTS

Material Facts 1-54 alleged in Broad Motion 2 are repeated in Appendix 2, along with
UCs concise responses thereto. Additional Material Facts 54-116 relied upon in support of this
Opposition are also set forth in Appendix 2.
IV.
8
9
ARGUMENT
In Broad Motion 2, Broad failed to meet its burden to prove that using a Type-II
CRISPR-Cas system in a eukaryotic cell renders Broads claims separately patentable.
10
11
12
A.
13
The Parties agree that all of Broads involved claims explicitly require operability of the
14
CRISPR-Cas9 system in a eukaryotic cell, and that none of UCs claims require performance of
15
the claimed method in a eukaryotic cell. See Broad Motion 2, at p. 1, ll. 21-22, p. 2, ll. 3-4.
16
Broad failed to show any other difference between the claims of the Parties. MF 55. Broad did
17
not meet its burden of proof to show that this difference renders the Parties claims separately
18
patentable.
19
Broads Sole Argument is That There Would Have Been No Reasonable

Expectation of Success in Using the Type-II CRISPR-Cas System of UCs
Claims in a Eukaryotic Cell
Broad Motion 2 does not dispute that a person of ordinary skill in the art was motivated
20
to use the Type-II CRISPR-Cas system of UCs claims in a eukaryotic cell. Broads Dr. Simons
21
acknowledged that there was a motivation to do so. MF 56; Ex. 1555, at p. 100, l. 17 - p. 102, l.
22
6. Rather, at page 13, lines 3-5, of Broad Motion 2, it is argued that the question presented is
23
whether a person of ordinary skill in the art would have had a reasonable expectation of success
24
in adapting the Type-II CRISPR-Cas system to function in a eukaryotic cell to cleave DNA.
25
And, at page 2, lines 13-16, of Broad Motion 2, it is argued that a person of ordinary skill would
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not have had any reasonable expectation that the Type-II CRISPR-Cas system would
successfully function in a eukaryotic cell.
Therefore, Broad Motion 2 rests entirely on an argument that there would have been no
reasonable expectation of success in using the Type-II CRISPR-Cas system of UCs claims in a
eukaryotic cell. Broad Motion 2 must be denied because the evidence overwhelmingly shows
that persons of ordinary skill in the art did have motivation, an expectation of success, and
indeed, reported success in using the Type-II CRISPR-Cas system of UCs claims in a
eukaryotic cell prior to Broads claims. Broad ignored, or failed to address, dispositive evidence
of obviousness. Consideration of the evidence shows that use of the Type-II CRISPR-Cas
10
system in eukaryotic cells would have been obvious prior to December 12, 2012. Ex. 1534,
11
5-10; Ex. 1535, 5-10.
12
B.
13
At page 20, lines 3-17, of Broad Motion 2, it is argued that UCs suggestion of
Broad Has the Burden of Proof
14
interference was inadequate. See also Broad Motion 2, at 13, ll. 8-11. At page 12, lines 24-27,
15
of Broad Motion 2, it is argued that to prove that a claim would have been obvious, a party must
16
demonstrate that the skilled artisan would have had a reasonable expectation of success. The
17
response is that Broad bears the burden of proof in this motion. See 37 C.F.R. 41.121(b).
18
Because the Declaration of this interference created a presumption that Broads claims were
19
obvious in view of UCs claims, and because the existence of a reasonable expectation of success
20
is the only basis on which Broad has challenged that presumption, Broad had the burden of
21
proving that there would have been no reasonable expectation of success in using a Type-II
22
CRISPR-Cas system in eukaryotic cells. Alarm.com v. Icontrol Networks, Inc., Interference No.
23
106,001, 2015 WL 1871503, at *24 (P.T.A.B. Mar. 31, 2015). That burden was not met.
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1
2
3
C.
Broad has completely ignored, or failed to rebut, overwhelming evidence of obviousness,
By Ignoring Prior Art, Broad Failed to Meet Its Burden of Proving that it
Would Not Have Been Obvious to Use UCs Type-II CRISPR-Cas System in
Eukaryotic Cells
and therefore failed to meet its burden to prove that there would have been no reasonable
expectation of success in using the Type-II CRISPR-Cas system of UCs claims in a eukaryotic
cell.
8
9
At page 19, lines 14-16, of Broad Motion 2, it is argued that none of the art available
prior to Broads disclosures would have led a person of ordinary skill in the art to have a
10
reasonable expectation of success of implementing a CRISPR-Cas system in eukaryotic cells. At
11
page 19, line 20 to page 20, line 2, of Broad Motion 2, it is argued that Dr. Simons reviewed the
12
relevant prior art and found no basis for the person of ordinary to have had any reasonable
13
expectation of successfully using CRISPR-Cas in a eukaryotic environment and that there was
14
no prior art that, combined with UCs involved claims, would have suggested or rendered the
15
eukaryotic subject matter of Broads involved claims obvious.
16
The response is that Broad and Dr. Simons failed to address relevant prior art, of which
17
they were aware, that conclusively shows that there was motivation, expectation of success, and
18
reports of success in using a CRISPR-Cas system in eukaryotic cells prior to Broads first
19
provisional application. Ex. 1534, 18-32; Ex. 1535, 18-32.
20
At page 19, lines 12-21, of Broad Motion 2, it is acknowledged that references prior to
21
December 12, 2012, the filing date of Broads first provisional are prior art to Broad. This is
22
correct as the earliest possible critical date. See, e.g., Alarm.com at *18-25 (assessing the state of
23
the art at the time of Broads filing for an interference-in-fact determination). However, Broad
24
made no showing that any of its involved claims actually have an effective filing date of
25
December 12, 2012, and thus failed to meet its burden to show that prior art after December 12,
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1
2012, is not also applicable. Nevertheless, for purposes of determining whether Broads claims
are obvious under the interference-in-fact test, the content of the prior art includes at least all
references prior to December 12, 2012. This includes Jinek 2012, in which UC publicly
disclosed the Type II CRISPR-Cas system that is recited in UCs involved claims, as well as the
contemporaneous reactions of those of ordinary skill in the art.
Such evidence, of which Broad was aware, but which Broad Motion 2 did not address,
can be found, for example, in U.S. Patent Application No. 61/717,324 (Ex. 1545) by Kim et al.
(the Kim Provisional) filed on October 23, 2012. 1 The Kim Provisional recognized that Jinek
2012 suggested using the Type-II CRISPR-Cas system in eukaryotes, stating:
10
11
12
13
14
Recently, Jinek et al. (2) elegantly demonstrated that a single-chain chimeric

RNA produced by fusing an essential portion of crRNA and tracrRNA could
replace the two RNAs in the Cas9 /RNA complex to form a functional
endonuclease, raising the possibility of using this system for genome editing in
cells and organisms.
15
Ex. 1545, at 7 (p. 1 of the specification); MF 58; Ex. 1534, 24; Ex. 1535, 24. The Kim
16
Provisional confirmed what Jinek 2012 had suggested, describing experiments using the Type-II
17
CRISPR-Cas system in eukaryotic cells, one of which is summarized as follows:
18
19
20
21
22
23
We co-transfected the Cas9-encoding plasmid, the guide RNA, and the RFP -GFP
reporter plasmid into human embryonic kidney (HEK) 293T cells, and found that
GFP- expressing cells were obtained only when the cells were co-transfected with
the Cas9 plasmid and the guide RNA (Fig. 2), demonstrating that [RNA -guided
endonucleases] could recognize and cleave the target DNA sequence in cultured
human cells.
24
Ex. 1545, at 9 (p. 3 of the specification); MF 59; Ex. 1534, 25; Ex. 1535, 25. This is
25
objective evidence that upon learning of UCs disclosure in Jinek 2012, and before Broads first
26
provisional, persons of ordinary skill in the art had an expectation that a Type-II CRISPR-Cas
1
The corresponding Published PCT Application WO 2014/065596 was cited in Broads patents.
See, e.g., Ex. 1010, cover sheet.
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1
system could be used in eukaryotic cells and were motivated by Jinek 2012 to do it. Ex. 1534,
26; Ex. 1535, 26. Indeed, on July 16, 2012, Jin-Soo Kim wrote to Jennifer Doudna, a UC co-
inventor, indicating that he had read Jinek 2012 with great interest and requesting biological
reagents. Ex. 1557. On October 3, 2012, Dr. Kim wrote to Drs. Doudna and Charpentier to
propose jointly publishing their results in mammalian cells. Ex. 1558.; see also Ex. 1598. He
noted that his group had been developing Cas9-based genome editing technology for the last
few months since we read your seminal Science paper. He also stated your Science paper
prompted us to start this project. Thus, it is clear that the experiments reported in the Kim
Provisional proceeded directly from UCs disclosure of the Type-II CRISPR-Cas system in Jinek
10
2012 and were completed within just a few months of its publication.
11
U.S. Patent Application Publication No. 20150322457 (Ex. 1599) (the Kim
12
Application), which is also not addressed in Broad Motion 2, claims the benefit of the Kim
13
Provisional and discloses and claims a Type-II CRISPR-Cas system for cleaving a target nucleic
14
acid sequence in a eukaryotic cell. Ex. 1599, [0013]; Claim 58; MF 60; Ex. 1534, 25; Ex.
15
1535, 25. The teachings of the Kim Application that were carried forward from the Kim
16
Provisional are prior art to Broad. The Kim Provisional and the Kim Application, neither of
17
which is addressed in Broad Motion 2, clearly show that Broads claims were obvious in view of
18
the Type-II CRISPR-Cas system recited in UCs claims.
19
Similarly, the prior art for purposes of the present inquiry also includes, under 35 U.S.C.
20
102(e), the disclosure of UCs 859 Application (Ex. 1001) with an effective date of subject
21
matter carried forward from UCs first provisional application, U.S. Patent Application No.
22
61/652,086, filed May 25, 2012 (Ex. 1003). MF 61; see, e.g., Ex. 1001, Claim 70; Ex. 1003,
23
Claim 61. Neither Broad Motion 2 nor Dr. Simons considered the teachings of UCs first
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provisional application. Ex. 1534, 27; Ex. 1535, 27. These teachings include expression of
a Type-II CRISPR-Cas system in eukaryotic cells (Ex. 1003, 124-129; Ex. 1001; 25, 103-
105; 119-120; 244-251; 277-282), or supplying a Cas9 site-directed modifying polypeptide to
eukaryotic cells as RNA or protein (Ex. 1003, 177-179; Ex. 1001; 287-289), for purposes
of targeting DNA contained therein (Ex. 1003, 165-177; 186-188, 216; Ex. 1001; 274-
275). MF62; Ex. 1534, 27; Ex. 1535, 27; see also Ex. 1003; Figs. 1-4; Ex. 1001; Figs. 1-3,
9.
8
9
Broad also ignored teachings in the prior art from before UCs Type-II CRISPR-Cas
system was fully disclosed, suggesting that CRISPR systems in general could be used in
10
eukaryotic cells. MFs 63, 64; Ex. 1534, 22; Ex. 1535, 22; Ex. 1161 (Sontheimer), at
11
0007. For example, Broad ignored the teachings of U.S. Patent Publication No. 2010/0076057,
12
filed September 23, 2009 by Sontheimer et al. Ex. 1161 (Sontheimer).2 Sontheimer shows
13
that, before the Type-II CRISPR-Cas system was first disclosed in a patent application by UC, it
14
had been suggested that CRISPR systems could be used in eukaryotic cells. Id. Although the
15
CRISPR-Cas system of Sontheimer is not a Type-II CRISPR-Cas system, and does not include a
16
Cas9 protein, Sontheimer demonstrates that persons of ordinary skill in the art were motivated to
17
use prokaryotic CRISPR systems in eukaryotic cells, were aware of conventional techniques
18
available to do so (including expression vectors, nuclear localization signals, and codon
19
optimization), and had an expectation of success in doing so. Id; see also Ex. 1161, at 0009,
20
0042, 0054, 0058, 0060.
Sontheimer was cited during the prosecution of U.S. Patent Application No. 14/054,414, which
issued as Broads involved 359 Patent. Ex. 1601, at 3-16.
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1
As this shows, Broad Motion 2 failed to address prior art that proves the obviousness of
using the Type-II CRISPR system in eukaryotic cells. Even for the art that was addressed, it
ignored relevant teachings therein, again failing to satisfy its burden of proof.
At page 3, line 15, to page 4, line 2, Broad Motion 2 argues that the experiments by
Senior Party inventors in Jinek 2012 (Ex. 1155) only contacted isolated components of a Type-II
CRISPR-Cas system with target DNA in a cell-free environment and did not disclose cleaving or
editing a target DNA molecule, or gene editing, and does not describe any experiments in any
type of cell, including eukaryotic cells.
The response is that Broads argument ignores the full content and context of Jinek 2012,
10
which in addition to showing the necessary and sufficient components of the Type-II CRISPR-
11
Cas system, also predicted the potential to exploit the system for RNA-programmable genome
12
editing months before Broad filed its first provisional application. Ex. 1155, Abstract; MFs 65,
13
66; Ex. 1534, 29-30; Ex. 1535, 29-30. Jinek 2012 further suggested the exciting
14
possibility of developing a simple and versatile RNA-directed system to generate dsDNA breaks
15
for genome targeting and editing. Ex. 1155, at 816, col. 2-3; Ex. 1534, 30; Ex. 1535, 30.
16
Jinek 2012 concludes with the following proposal:
17
18
19
20
21
Zinc-finger nucleases and transcription-activatorlike effector nucleases have

attracted considerable interest as artificial enzymes engineered to manipulate
genomes (3538).We propose an alternative methodology based on RNAprogrammed Cas9 that could offer considerable potential for gene-targeting and
genome-editing applications.
22
Ex. 1155, at 820, col. 3; MF 67; Ex. 1534, 30; Ex. 1535, 30. Zinc-finger nucleases
23
(ZFNs) and transcription-activatorlike effector nucleases (TALENs) were, at the time of
24
Jinek 2012, the state of the art for DNA cleavage and editing in eukaryotic cells. MFs 68, 69;
25
Ex. 1534, 31; Ex. 1535, 31. Thus, the proposal in Jinek 2012 to replace ZFNs and TALENS
26
with the programmable Type-II CRISPR-Cas system would have been understood by those of
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ordinary skill in the art to be an explicit proposal to use the Type-II CRISPR-Cas system for
eukaryotic gene editing. Id. This understanding is shown by commentary that accompanied the
publication of Jinek 2012. Ex. 1534, 32; Ex. 1535, 32. There, Stan Brouns of Wageningen
University wrote:
5
6
7
8
9
10
11
12
13
14
15
16
Jinek et al. realized that a highly specific, customizable RNA-directed DNA

nuclease could be useful to edit whole genomes. Based on the 20-nucleotide guide
section of the crRNA, the enzyme could theoretically introduce breaks at unique
sites in any eukaryotic genome. . . . Introducing DNA breaks at desired loci using
just Cas9 and a chimeric crRNA would be a substantial improvement over
existing gene-targeting technologies, such as zinc finger nucleases and
transcription activatorlike effector nucleases, as these require protein
engineering for every new target locus (10). Efficient gene repair strategies in
cells from patients, and the reintroduction of repaired cells, could become
increasingly important for treating many genetic disorders.
Ex. 1471 (emphasis added); MF 71.
Brouns comments, which are also prior art to Broad, demonstrate not just a
17
contemporaneous expectation by someone in the art that the Type-II CRISPR-Cas system would
18
cleave DNA in eukaryotic cells, but a prediction that the Type-II CRISPR-Cas system could
19
become an important tool for treating genetic disorders. Ex. 1534, 32; Ex. 1535, 32. This is
20
not the commentary of a person who thought that there was no reasonable expectation of
21
successfully using the Type-II CRISPR-Cas system in eukaryotic cells. Id. Jinek 2012 and the
22
commentary accompanying its publication demonstrate a high expectation of success that
23
objectively contradict the sole argument on which Broad Motion 2 rests. In ignoring this
24
evidence, Broad further failed to meet its burden of proof.
25
26
27
D.
28
At page 2, lines 16-20, page 5, lines 4-26, page 11, lines 21-23, page 18, lines 6-25, of
29
Broad Motion 2, it is argued that contemporaneous evidence shows that skilled artisans would
Contemporaneous Evidence Demonstrates Motivation and Reasonable

Expectation of Success in Using the Type-II CRISPR-Cas System in
Eukaryotes
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not have reasonably predicted that CRISPR systems would function in eukaryotes. At page 19,
lines 1-7, of Broad Motion 2, it is argued that as of 2012, when Broad was doing its initial work,
applying a Type-II CRISPR-Cas system in eukaryotic cells required overcoming uncertainty
regarding whether such a system might function in eukaryotic cells.
The response is that the contemporaneous evidence proves that as soon the system recited
in UCs claims became known, Broad was just one of many groups that quickly confirmed, using
conventional techniques, that the Type II CRISPR-Cas system could be readily used in
eukaryotic cells. MFs 71-83; Ex. 1534, 33-38, 62-74; Ex. 1535, 33-38, 62-74. Any
speculative uncertainties argued in Broad Motion 2 were clearly not sufficient to dissuade people
10
of ordinary skill in the art from using the Type-II CRISPR-Cas system disclosed by UC in Jinek
11
2012 in many different types of eukaryotic cells. Id.
12
13
14
15
1.
Multiple Researchers in the Art Had an Expectation of Success in

Adapting UCs System to Eukaryotic Cells Before Broad Published
Its Results
As the saying goes, actions speak louder than words. Before Broad filed its first
16
provisional application on December 12, 2012, at least two other independent groups had
17
submitted manuscripts for peer review and publication that demonstrated use of the Type-II
18
CRISPR-Cas system in eukaryotic cells, and more groups submitted manuscripts within a week
19
of Broads filing. MF 71, 72; Ex. 1534, 63; Ex. 1535, 63. Manuscripts by Mali et al. (Ex.
20
1056), Cho et al. (Ex. 1059), Jinek et al. (Ex. 1057), and Hwang et al. (Exs. 1058, 1554) were
21
submitted between October 26, 2012, and December 18, 2012, prior to, or contemporaneously
22
with, Broads filing of its first provisional application. Id. Each manuscript was published in
23
January 2013, contemporaneously with Broads first publication. MF 73; Ex. 1534, 63-64;
24
Ex. 1535, 63-64. Each group specifically cited Jinek 2012 as a basis of their work. MF 74;
25
Ex. 1534, 72; Ex. 1535, 72. The table below summarizes manuscript submissions that
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1
occurred through December 2012 showing use of a Type II CRISPR-Cas system in eukaryotic
cells. Ex. 1534, 63, Fig.; Ex. 1535, 63, Fig. 1.

Author
(Exhibit No.)
Mali et al.
(Ex. 1056)
Cho et al.
(Ex. 1059)
Jinek et al.
(Ex. 1057)
Hwang et al.
(Exs. 1058, 1554)
3
4
Submission Date
Cell Type
Publishing Journal
October 26, 2012
Human
SCIENCE
November 20, 2012
Human
NATURE BIOTECHNOLOGY
December 15, 2012
Human
ELIFE
December 18, 2012
Zebrafish
NATURE BIOTECHNOLOGY
Each of these publications was from a different independent research group. MF 75; Ex.
1534, 62-72; Ex. 1535, 62-72. These groups would not have undertaken the use of UCs
Type-II CRISPR-Cas system in eukaryotic cells unless there was sufficient motivation and
expectation of success. George Church and Jin Soo Kim, lead investigators on the Mali and Cho
papers, respectively, each independently contacted Drs. Doudna and Charpentier prior to
December 12, 2012, and stated that Jinek 2012 had motivated their work. MF 76; see Exs.
10
1558-1560. George Church has publicly stated that the work of Mali et al. was independent of
11
the Zhang lab of Broad. MF 77; Ex. 1534, 67; Ex. 1535, 67. The research following UCs
12
disclosure was performed in a variety of eukaryotic cells: human (Cho et al., Mali et al., Jinek et
13
al.), zebrafish (Hwang et al.; Shen et al.), mouse (Shen et al.), and yeast (DiCarlo et al.) cells.
14
MF 78; Ex. 1534, 62-72 and Appendix; Ex. 1535, 62-72 and Appendix; Exs. 1056-1060,
15
1372. All these groups would not have undertaken the use of UCs Type-II CRISPR-Cas system
16
in eukaryotic cells without both motivation and an expectation of success.
17
None of these demonstrations of the Type-II CRISPR-Cas system in eukaryotic cells
18
required any unusual reagents or techniques, but rather used conventional techniques in
19
conventional ways to produce predictable and expected results. Ex. 1534, 70 and Appendix;
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1
Ex. 1535, 70 and Appendix; Ex. 1055, at Fig. 1B; Ex. 1056, at Fig. 1A; Ex. 1059 at
Supplementary Methods 2; Ex. 1058, at Methods; Ex. 1060, at 720. All of these groups used
well-known and conventional promoters. Id. The Hwang and Shen groups did not use vectors,
but rather used the well-known technique of directly injecting the system components as RNA
into cells. Ex. 1534, 75 and Appendix; Ex. 1535, 75 and Appendix; Exs. 1058, 1060. Cho et
al. used a vector to express Cas9, but delivered guide RNA directly. Ex. 1534, 75 and
Appendix; Ex. 1535, 75 and Appendix; Ex. 1059.
8
9
This is further objective evidence that the disclosure of the Type-II CRISPR-Cas system
by UC was all that was required to render obvious the use of the system in any eukaryotic cell.
10
See KSR Intl Co. v. Teleflex Inc., 127 S.Ct. 1727, 1739 (2007) (finding obviousness where [t]he
11
combination of familiar elements according to known methods . . . does no more than yield
12
predictable results).
13
14
15
2.
Like All the Others, Broad Merely Made Obvious Modifications to

UCs Invention and Only After UCs Disclosure
Listed inventors of Broad have made admissions showing that Broad was merely one of
16
the many groups motivated, with an expectation of success, to move the Type-II CRISPR-Cas
17
system into eukaryotic cells after seeing the system fully elucidated in Jinek 2012. For example,
18
Shuailiang Lin, a former Zhang laboratory member and a listed co-inventor on Broads fist
19
provisional patent application (see Ex. 1602), admitted to Dr. Doudna on February 28, 2015, that
20
the Zhang laboratory did not work [the use of the Type-II CRISPR-Cas system in eukaryotic
21
cells] out before seeing [the Jinek 2012] paper. Ex. 1475, Ex. 1571; MF 80. Below is an
22
excerpt from that email:
- 12 -
1
2
See Ex. 1475. As Shuailiang Lins email admits, Feng Zhang and Le Cong jumped to the
project to use the Type-II CRISPR-Cas system in eukaryotic cells only after seeing UCs
disclosure of the system in Jinek 2012 in June of that year. Id.; MF 81.
Shuailiang Lins statement is consistent with the admissions of another Zhang lab
member, Fei-Ann Ran, who is a co-author of the Cong publication and one of Broads named
inventors. Describing the developments preceding the Zhang laboratorys eukaryotic cell
experiments, she wrote:
9
10
11
12
13
When we started working on this, the tracrRNA hadnt been discovered yet. . . .
At that time, two other developments also emerged (1) you can fuse the spacer
and the repeat and the tracrRNA into a single chimeric RNA; and (2) you can use
a single chimeric RNA and Cas9 to program the cleavage of DNA targets in an in
vitro cell-free lysis reaction. We built upon these exciting discoveries.
14
Ex. 1561, at pp. 71-73; MF 82. She admits that they built on the exciting discoveries that
15
were first disclosed in Jinek 2012, which she references in her paper. Fei-Ann Ran further
16
explained that they used only two adaptationsboth conventionalto move UCs Type-II
17
CRISPR-Cas system into mammalian cells:
18
19
20
21
obviously, bacteria dont have nuclei, whereas mammalian and other eukaryotic
cells do, and so we tagged NLS (nuclear localization signal) sequences to Cas9
and also codon-optimized it for better eukaryotic expression. By doing this, we
were successful in moving the Cas9 enzyme into mammalian nuclei.
22
Ex. 1561, at p. 73; MF 83. Dr. Simons acknowledged that both NLSs and codon optimization
23
used by the Zhang lab were conventional in 2012. MF 88; see Ex. 1555, at p. 97, ll. 19-22, p.
24
132, l. 11 p. 134, l. 20. These admissions by Zhang lab members show that the invention
- 13 -

1
that Broad argues is separately patentable from UCs claims depended entirely on UCs
disclosure of the necessary components of the Type-II CRISPR-Cas system that are recited in
UCs claims. Once the functional system had been disclosed by UC, using the system in
eukaryotic cells was quickly and easily accomplished using only obvious and conventional
techniques.
6
7
8
9
3.
The Contemporaneous Quotations Cited by Broad Further Evidence

Obviousness When Placed in Context
Broad Motion 2 cites selected quotes to argue the alleged nonobviousness of using the
Type-II CRISPR-Cas system in eukaryotic cells. For example, at page 5, lines 4-17, Broad
10
quotes Dr. Dana Carroll as stating that [t]here is no guarantee that Cas9 will work effectively on
11
a chromatin target or that the required DNARNA hybrid can be stabilized in that context. See
12
also Broad Motion 2, at p. 18, ll. 6-12. Likewise, Broad quoted Dr. Carroll as saying that
13
[o]nly attempts to apply the system in eukaryotes will address these concerns.
14
The response is that when Broads selective quotations are placed in context, they
15
actually demonstrate the opposite. Ex. 1534, 34-36 and 68-70; Ex. 1535, 34-36 and 68-70.
16
Broad omitted, and failed to address, the more salient points of Dr. Carrolls commentary. Broad
17
omitted the fact that in the same article, Dr. Carroll explained how Jinek 2012 suggested use of
18
the Type-II CRISPR-Cas system in higher organisms.
19
20
21
The authors [of Jinek 2012] make the bold prediction that this system can
potentially be used in place of ZFNs or TALENs for targeted genomic cleavage in
higher organisms. Lets think about how this might work.
22
Ex. 1152, at 1659; MF 84; Ex. 1534, 34; Ex. 1535, 34. And, particularly, Broad omitted
23
from its quotation the most salient comment to the present inquirywhere Dr. Carroll, with
24
regard to the use of the Type-II CRISPR-Cas system in eukaryotic cells, states: Whether the
25
CRISPR system will provide the next-next generation of targetable cleavage reagents remains to
- 14 -

1
be seen, but it is clearly well worth a try. Stay tuned. Ex. 1152, at 1660 (emphasis added); MF
85; Ex. 1534, 34; Ex. 1535, 34. When the full content of Dr. Carrolls contemporaneous
comments, which are prior art to Broads claims, is considered, he clearly recognized the
obviousness of moving the system into eukaryotic cells and suggested doing so. Ex. 1534,
34-36; Ex. 1535, 34-36. And, in encouraging the readers to stay tuned, Dr. Carrolls
comments demonstrate an expectation that it would be done. Id.
At page 18, lines 20-21, of Broad Motion 2, it is argued that Marraffini was quoted as
saying that [i]ts not trivial to make CRISPR/Cas systems work in eukaryotic cells One thing
is to have them in silico and have a sequence and another thing is to do the experiments and
10
make it work. A first response is that scientific experimentation need not be trivial for there
11
to be a reasonable expectation of success. That is not the law. Further, the purported quotation
12
is not referring to the use of known Cas9 proteins in eukaryotic cells, but is referring to proteins
13
that have been identified only by sequence. Broads misconstruction of the quote is contradicted
14
by the many other contemporaneous comments. Id. As noted above, before anyone had even
15
reported trying the Type-II CRISPR-Cas system in eukaryotic cells, one commenter predicted
16
the [Cas9] enzyme could theoretically introduce breaks at unique sites in any eukaryotic
17
genome . . . [and] could become increasingly important for treating many genetic disorders. Ex.
18
1471; MF 70; Ex. 1534, 32; Ex. 1535, 32. Another author, reviewing the research that
19
followed Jinek 2012, stated that [i]t was immediately obvious that [the Type-II CRISPR-Cas]
20
system might be repurposed for genome engineering, similar to ZFNs and TALENs. MF 86;
21
see Ex. 1473, at 306; Ex. 1534, 36; Ex. 1535, 36. Thus, the contemporaneous statements of
22
those of ordinary skill in the art actually demonstrated an expectation that the Type-II CRISPR
23
system could be successfully employed in eukaryotic cells.
- 15 -

1
2
3
E.
At page 20, lines 5-7, of Broad Motion 2, it is argued that UCs Suggestion of
Broad Fails to Address Extensive Evidence that the State of the Art
Rendered Use of the Type-II CRISPR-Cas System in Eukaryotic Cells
Obvious Once the System Was Described
Interference simply argued that it would have been routine for one of ordinary skill in the art to
use known methods and materials to apply the Type-II CRISPR Cas system to eukaryotic cells.
The response is that Broads argument ignores, simplifies, and glosses over substantial evidence
that use of prokaryotic proteins, including DNA-targeting prokaryotic proteins, was well
established using methods that had become routine. MFs 87-93; Ex. 1534, 52-61 and 78-87;
10
Ex. 1535, 52-61 and 78-87.
11
12
13
1.
Broad Failed to Address the Fact that All of the Techniques Used to
Introduce the Type-II CRISPR-Cas System into Eukaryotic Cells
Were Routine
14
Persons of ordinary skill in the art would have had a reasonable expectation of success in
15
using UCs Type-II CRISPR-Cas system in eukaryotic cells because all the techniques one might
16
use to practice the methods of UCs claims in eukaryotic cells were well-known and routinely
17
used in the art. MFs 87-93; Ex. 1534, 39-51; Ex. 1535, 39-51. Dr. Simons admitted
18
that a person of ordinary skill in the art would be capable of performing the conventional
19
methods, and that all of the techniques one might use to apply Type-II CRISPR-Cas in
20
eukaryotic cells, including those taught in Broads own patents and application, were
21
conventional at the time. MF 88; see Ex. 1555, at p. 97, ll. 19-22, p. 134, ll. 5-20 (techniques
22
described as conventional), p. 146-153 (specifically referring to techniques described in
23
Broads provisional application, Ex. 2101); Ex. 1534, 51; Ex. 1535, 51.
24
Indeed, methods for introducing a nucleic acid into a eukaryotic cell had been well-
25
known for over 30 years. MF 89; see, e.g., Exs. 1040; 1200; 1248; 1233; 1260; 1307; Ex. 1534,
26
44; Ex. 1535, 44. It was well-known that a protein or nucleic acid, including those of
- 16 -

1
prokaryotic origin, could be expressed in a eukaryotic cell using an expression vector, including
viral vectors. MF 90; see, e.g., Exs. 1031; 1039; 1194; 1203; 1218; 1229; 1332; 1337; 1353;
Ex. 1534, 44; Ex. 1535, 4. And, in fact, these well-known and routinely used viral vectors
had been previously used to express prokaryotic DNA-targeting proteins in eukaryotic cells.
See, e.g., Ex. 1285; Ex. 1283; Ex. 1534, 44; Ex. 1535, 44. It was well-understood that
heterologous expressed proteins could be targeted to the nucleus, where genomic DNA is located
in eukaryotic cells, and methods, including the use of one or more nuclear localization sequences
(NLSs), had been routinely used for decades. MF 91; see, e.g., Exs. 1029; 1235; 1236; 1319; Ex.
1534, 45; Ex. 1535, 45. One of ordinary skill in the art understood how to increase
10
expression of prokaryotic proteins in eukaryotic cells by codon optimization. MF 92; see, e.g.,
11
Ex. 1030; Ex. 1534, 45; Ex. 1535, 45; see also Ex. 1315; Ex. 1576, at 796-98. However,
12
neither modification was necessary to achieve Type-II CRISPR-Cas mediated cleavage of target
13
DNA in a eukaryotic cell. See Ex. 1058; Ex. 1060. Ex. 1534, 46; Ex. 1535, 46. Directly
14
injecting a pre-assembled prokaryotic protein/RNA complexes into eukaryotic cells, and thereby
15
avoiding the need to use expression vectors, had also been used to affect genetic modifications.
16
MF 93; see, e.g., Ex. 1293, at 12; Ex. 1534, 46; Ex. 1535, 46.
17
Thus, the techniques one might use to apply a Type-II CRISPR system in eukaryotic
18
cells, including those that are taught in Broads patents and application, were conventional at the
19
time Broads first provisional was filed, providing those of ordinary skill in the art with an
20
expectation of success in using the Type-II CRISPR system in eukaryotic cells.
21
22
23
2.
Examples of Successful Use of Prokaryotic DNA-Targeting Proteins in

Eukaryotic Cells Provided a Reasonable Expectation that the
Prokaryotic DNA-Targeting Protein Cas9 Would Likewise Function
24
These conventional methods for expressing functional prokaryotic proteins and nucleic
25
acids in eukaryotic cells were accompanied by a long history of analogous prokaryotic systems
- 17 -

1
being used in eukaryotic cells. MFs 94-102; Ex. 1534, 52-61; Ex. 1535, 52-61. By 2012,
therefore, a person of ordinary skill in the art would have expected that most prokaryotic proteins
of interest could be routinely used in eukaryotic cells. Id.
For example, the prokaryotic Cre-Lox site-specific recombination system had been used
successfully in eukaryotic cells since the late 1980s. MFs 95, 100, 101; Ex. 1534, 54-55; Ex.
1535, 54-55; see Ex. 1335 (Sauer); Ex. 1336; Ex. 1595 (stating [t]he number of
transgenic mouse lines expressing Cre recombinase with different specificities has steadily
increased in the past 15 years and now has surpassed 500, and [t]he Cre/loxP system has
proven to be the most efficient [site-specific recombination system] in mammalian cells.); Ex.
10
1594; Ex. 1596. The prokaryotic EcoRI restriction endonuclease had also been used in
11
eukaryotic cells since the late 1980s. MF 96; Ex. 1534, 56; Ex. 1535, 56; see Ex. 1302
12
(Morgan et al.). The prokaryotic RecA protein had been used successfully in eukaryotic cells
13
from at least the mid-1990s. MF 97, 102; Ex. 1534, 57-59; Ex. 1535, 57-59; see Ex. 1329
14
(Reiss et al.). The C31 recombinase from Streptomyces lividans was well-known to display
15
activity in mammalian cells. MF 98; Ex. 1534, 60; Ex. 1535, 60; see Ex. 1327 (Raymond
16
and Soriano). DNA-targeting ZFN and TALEN systems, which were the state of the art in
17
2012 and contain the prokaryotic Fok1 endonuclease domain, would have also provided a person
18
of ordinary skill in the art with an expectation that the analogous Type-II CRISPR-Cas system
19
could be used in eukaryotic cells successfully. MF 99; Ex. 1534, 52; Ex. 1535, 52. There
20
are countless examples of other types of prokaryotic proteins that had been successfully
21
employed in eukaryotic cells long before 2012, such as bacterial antibiotic resistance genes. Ex.
22
1534, 53; Ex. 1535, 53; see, e.g., Ex. 1581; Ex. 1310; Ex. 1248; Ex. 1575; Ex. 1580; Ex.
23
1574. All of these prior successes would have given a person of ordinary skill in the art a
- 18 -

1
reasonable expectation of success in using the Type-II CRISPR-Cas system in eukaryotic cells.
MFs 94-102; Ex. 1534, 52-61; Ex. 1535, 52-61.
3
4
3.
Broads Arguments are Unsupported and Contradicted by Evidence
At page 20, line 18 to page 21, line 6, of Broad Motion 2, it is argued that ZFNs and
TALENs are not relevant because these proteins contain elements derived from eukaryotes. The
response is that Broads argument ignores the fact that ZFNs and TALENs utilize the prokaryotic
FokI restriction endonuclease domains to cleave eukaryotic target DNA. MF 99; Ex. 1534, 52;
Ex. 1535, 52. Thus, the DNA cleaving domain of these systems is prokaryotic. Id. The well-
known application of ZFNs and TALENs for eukaryotic genome editing would have provided a
10
person of ordinary skill in the art with a reasonable expectation that another heterologous,
11
prokaryotic DNA-cutting protein, such as Cas9, could also be successfully used in eukaryotic
12
cells. Id.
13
At page 21, line 19 to page 22, line 3, of Broad Motion 2, it is argued that Sauer 1987
14
(Ex. 1335) and Sauer 1988 (Ex. 1336) show that there once was some doubt as to whether the
15
Cre protein could access a DNA site in a chromatin structure. The response is that any doubt
16
that a prokaryotic DNA-targeting protein could work in a chromatin context was laid to rest by
17
Sauer in 1987more than 25 years before Broads first provisional application. MFs 95, 100,
18
101; Ex. 1534, 54-55; Ex. 1535, 54-55. Sauers results demonstrate that a procaryotic
19
recombinase can enter a eucaryotic nucleus and, moreover, that the ability of the Cre
20
recombinase to perform precise recombination events on the chromosomes of S. cerevisiae is
21
unimpaired by chromatin structure. Id.; see also Ex. 1335, abstract (emphasis added). Dr.
22
Simons acknowledged that Sauer demonstrated the use of Cre-Lox recombinase in eukaryotic
23
cells. Ex. 1556, at 234, l. 21-235, l. 14.
24
At page 22, lines 4-9, of Broad Motion 2, it is argued that because authors of Reiss et al.
- 19 -

1
were awarded a patent for use of the prokaryotic DNA cutting protein (RecA) in eukaryotic cells,
it should be recognized that their success was unpredictable. The response is that even if the use
of RecA in eukaryotes was surprising in 1996, such a successful adaptation could no longer be
considered surprising16 years laterin 2012. MF 102; Ex. 1534, 57-59; Ex. 1535, 57-
59. Reiss et al. demonstrated that that the prokaryotic recombination protein RecA itself is
capable of interacting with genomic homologous DNA in somatic plant cells. Id.; see also Ex.
1329, Abstract; Ex. 1556, at 240, ll.11-14. The successful use of RecA, in 1996, would have
provided a reason to expect that another prokaryotic DNA-targeting protein, such as Cas9, would
also work in eukaryotic cells in 2012. Id.
10
At page 22, lines 9-13, of Broad Motion 2, it is argued that because a protein homologous
11
to RecA can be found in eukaryotes, the successful use of prokaryotic RecA in eukaryotes would
12
not provide a basis for a reasonable expectation of success in the adaptation of Cas9. The
13
response is that the prokaryotic RecA was known to be functional in eukaryotes. MF 97; Ex.
14
1534, 59; Ex. 1535, 59. This fact demonstrates that it was known that a protein that was
15
naturally adapted for prokaryotes was nevertheless capable of editing eukaryotic DNA. Id.
16
17
18
19
4.
Broads Arguments of Non-Specific Differences Between Prokaryotes

and Eukaryotes Do Not Dispel the Evidence of Reasonable
Expectation of Success
The successful use of the prokaryotic Cre, EcoRI, RecA, and C31 DNA-targeting
20
proteins in eukaryotes also contradicts another of Broads arguments. At page 4, line 17 to page
21
5, line 3, of Broad Motion 2, it is argued that those of ordinary skill in 2012 would have
22
considered the use of a Type-II CRISPR-Cas system in eukaryotic cells to be unpredictable
23
because of the many differences between the cellular environments of prokaryotic and eukaryotic
24
cells. At page 6, lines 5-17, of Broad Motion 2, it is argued that a listing of several more
25
purported differences between prokaryotic and eukaryotic cells would have caused a person of
- 20 -

1
2
ordinary skill in the art to have no reasonable expectation of success.

The response is that the many examples of prokaryotic DNA-targeting proteins that were
known to function in eukaryotic cells, discussed in detail above, demonstrate that persons of
ordinary skill in the art would have recognized that none of these differences had been shown to
prevent prokaryotic DNA-targeting proteins from functioning in eukaryotic cells. MFs 94-12;
Ex. 1534, 52-61; Ex. 1535, 52-61. On cross examination, Dr. Simons admitted that he has
not cited any evidence that any of the differences between prokaryotic and eukaryotic cells has
actually presented any specific difficulty in using the Type-II CRISPR-Cas system and methods
in eukaryotic cells. MF 103; see Ex. 1555, p. 179, l. 8-p. p. 180, l. 9, p. 192, l. 10 p. 193, l. 16,
10
p. 202, l. 2 22,. p. 203, ll. 16-21. Persons of ordinary skill in the art would not, therefore, have
11
viewed any of these as a likely impediment to the use of the Type-II CRISPR system in
12
eukaryotic cells. Ex. 1534, 93-109; Ex. 1535, 93-109.
13
At page 6, lines 18-22, of Broad Motion 2, it is argued that because prokaryotic proteins
14
like Cas9 evolved in the context of prokaryotic cells and their protein folding environment, the
15
folding of a Cas9 protein in a eukaryotic cell was unpredictable. See also Broad Motion 2, at p.
16
15, ll. 14-16. In addition to the fact that the other prokaryotic proteins discussed above were
17
each demonstrated to properly fold so as to be functional in eukaryotic cells, the response is that
18
Dr. Simons admitted on cross-examination that he did not have any evidence that misfolding
19
proteins was actually a factor or an impediment to the successful use of Cas9 systems in
20
eukaryotic cells. MF 104; Ex. 1555, at 179, ll. 14-19. Moreover, because it was known that
21
functional proteins could be injected into eukaryotic cells, a person of ordinary skill in the art
22
would have understood that the ability to fold in eukaryotic cells was not a requirement for Type-
23
II CRISPR-Cas systems to be used in eukaryotic cells. See Ex. 1555, at 180-183; Ex. 1534,
- 21 -

1
78-79; Ex. 1535, 78-79. Thus, there is no evidence that a person of ordinary skill in the art
would have considered protein folding to be an unpredictable impediment to using Cas9 in a
eukaryotic cell. Ex. 1534, 78-79; Ex. 1535, 78-79. Indeed, these examples show the
insignificance of all of the non-specific, speculative unknowns cited in Broad Motion 2.
At page 5, line 26 to page 6, line 3, of Broad Motion 2, it is argued that eukaryotic
chromosomes are composed of chromatin, a complex and tightly-packed structure. See also
Broad Motion 2, at p. 7, ll. 16-23, p. 15, ll. 6-13. The response is, as noted above, several
examples were known in the art showing that prokaryotic DNA-targeting proteins could access
and act on eukaryotic chromosomes composed of chromatin. MFs 94-102; Ex. 1534, 55 and
10
95-97; Ex. 1535, 55 and 95-97. For example Sauer showed that Cre recombinase . . . is
11
unimpaired by chromatin structure. Ex. 1335, abstract; Ex. 1534, 55; Ex. 1535, 55. None
12
of the other prokaryotic DNA-targeting proteins mentioned above (EcoRI, RecA, C31) were
13
apparently impeded by chromatic structure. Ex. 1534, 95-97; Ex. 1535, 95-97. Moreover,
14
persons of ordinary skill in the art would have recognized that eukaryotic DNA is not fixedly
15
bound in chromatin, it was known to be a dynamic system, constantly rearranging and exposing
16
different areas of DNA. MF 105; Ex. 1534, 96; Ex. 1535, 96. There are times in the cell
17
cycle when nuclear DNA must be made accessible, for example during stages of cell division
18
when the entire genome must be copied. Id.; see also Ex. 1556, p. 232, l. 7 p. 233, l. 6.
19
Further, as Dr. Simons admitted, the potential target DNA in a eukaryotic cell is not limited to
20
chromatin bound genomic DNA. MF 106; See, e.g., Ex. 1556, p. 229, l. 16-p. 230, l. 15.
21
Plasmids and pathogenic DNA that are not bound to chromatin could also be targets of the Type-
22
II CRISPR-Cas system. Id.
23
As for the gene expression machinery cited at page 6, in lines 5-17, of Broad Motion 2, as
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1
shown above, the use of techniques to optimize expression of prokaryotic proteins, e.g., codon
optimization, was well-known and routine. MF 92; Ex. 1534, 98; Ex. 1535, 98; see, e.g.,
Exs. 1031; 1039; 1194; 1203; 1218; 1229; 1332; 1337; 1353. As to cellular
compartmentalization, the targeting of proteins (including prokaryotic proteins) to the nucleus of
eukaryotes was also routine in the art long before UC filed its first provisional application, as
shown in detail above. MF 91; Ex. 1534, 99; Ex. 1535, 99; see, e.g., Exs. 1029; 1235; 1236;
1319. As for intracellular metal ion concentrations (see also Broad Motion 2, at p. 17, ll. 13-18),
intracellular pH, modifications to DNA and RNA, nucleases and other molecules present in
eukaryotic cells, many examples were known in the art demonstrating that prokaryotic DNA
10
modifying enzymes could function in the conditions of eukaryotic cells, as shown in detail
11
above. Ex. 1534, 100; Ex. 1535, 100; see, e.g., Ex. 1335, Ex. 1336; Ex. 1302, Ex. 1329,
12
Ex. 1327. Likewise, examples were known in the art of RNA molecules that could be expressed
13
in eukaryotic cells and functionally interact with proteins. MF 107; see, e.g., Ex. 1229. As to
14
temporal and spatial requirements cited at page 6, line 22 to page 7, line 6, of Broad Motion 2, it
15
was routine in the art to target proteins (including prokaryotic DNA-targeting enzymes) to the
16
nucleus of eukaryotic cells, to express RNA in the nucleus of eukaryotic cells, and for RNA
17
expressed in the nucleus of eukaryotic cells to functionally interact with targets inside the
18
nucleus. MF 91; Ex. 1534, 102; Ex. 1535, 102; see, e.g., Ex. 1329, at 3094 (expressing a
19
RecA construct coding for a fusion protein of a nuclear targeting sequence). Researchers had
20
also directly injected a pre-assembled prokaryotic protein/RNA complex into eukaryotic cells to
21
cause genome modification in those cells, thereby obviating any alleged need for spatial and
22
temporal coordination within the cell. MF 93; Ex. 1534, 102; Ex. 1535, 102; see, e.g., Ex.
23
1293, at 12.
- 23 -

1
At page 7, lines 7-15, of Broad Motion 2, it is argued that it could not have been
predicted whether the components of a Type-II CRISPR-Cas system would be sufficiently stable
to interact and function in eukaryotic cells because of un-named intracellular degradation
pathways and speculative cellular toxicity. See also Broad Motion 2, at p. 15, l. 17 - p. 17, l. 2.
The response is that, as shown above, examples of the successful expression of prokaryotic
proteins and RNA in eukaryotic cells were well-known prior to the filing of UCs first
provisional application. Researchers had also directly injected a pre-assembled prokaryotic
protein/RNA complex into eukaryotic cells to cause genome modification in those cells, thereby
avoiding the need to use expression vectors and rely on the cells protein production machinery.
10
MF93; Ex. 1534, 104; Ex. 1535, 104; see, e.g Ex. 1293, at 12.
11
None of Broads arguments relating to alleged, non-specific differences between
12
prokaryotic and eukaryotic cells were supported by any relevant examples where these alleged
13
issues actually caused any prokaryotic system to fail in a eukaryotic environment.
14
15
16
17
5.
Broads Non-Analogous Examples Would Not Dispel the Expectation

of Successfully Using the Type-II CRISPR-Cas System in Eukaryotic
Cells
At page 21, lines 7-11, of Broad Motion 2, it is argued that UCs Suggestion of
18
Interference did not point to any RNA-based systems that were successfully transferred to
19
eukaryotes. In making this argument, Broad appears to imply that the foregoing examples can be
20
dismissed because they are not RNA-based systems. However, the arguments at page 16, line
21
3 to page 17, line 18, of Broad Motion 2, directed to RNA-based systems are not directed to
22
catalytic proteins like Cas9. MF 109; Ex. 1534, 88-92; Ex. 1535, 88-92.
23
Cre, EcoRI, RecA, and C31 demonstrate that persons of ordinary skill in the art would
24
have been aware of successful uses of prokaryotic DNA-targeting proteins in eukaryotic cells
25
prior to UCs disclosure. Like each of these examples, Cas9 is a DNA-targeting protein. MF
- 24 -

1
109; Ex. 1534, 88; Ex. 1535, 88. Indeed, Cas9 is an endonuclease protein having a similar
DNA cleaving function as EcoRI. Ex. 1534, 81; Ex. 1535, 81.
At page 16, line 3 to page 17, line 12, of Broad Motion 2, it is argued that a person skilled
in the art would have been aware of obstacles previously encountered in attempts to transfer
prokaryotic, RNA-based riboswitches, ribozymes, and self-splicing Group II introns.
The response is that, Dr. Simons admitted on cross examination that each of the
ribozymes, riboswitches, and self-splicing introns that he cited as examples, were actually shown
to function in eukaryotic cells in the references that he himself cited. MF 108; see Ex. 1556, p.
216, ll. 19-20, p. 219, l. 21 222, l. 18, p. 225, ll. 5-13. Numerous other references confirm the
10
successful use of Group-II introns, ribozymes, and riboswitches in eukaryotic cells. See, e.g.,
11
Ex. 1577; Ex. 1583; Ex. 1582; Ex. 1534, 84; Ex. 1535, 84. Since none of Dr. Simons
12
examples of RNA based systems actually failed to work in eukaryotic cells, Broad did not
13
present evidence of any prokaryotic system that failed to work in eukaryotic cells. MF 108; Ex.
14
1534, 83-87; Ex. 1535, 83-87.
15
Moreover, the activity of riboswitches, ribozymes, and self-splicing introns comprised of
16
RNA would simply not be relevant to the potential activity of the Cas9 protein. MF 110; Ex.
17
1534, 88-92; Ex. 1535, 88-92. Riboswitches, ribozymes, and self-slicing introns are not
18
proteins like Cas9. MF 109; Ex. 1534, 88-89; Ex. 1535, 88-89. Thus, a person of
19
ordinary skill would not have considered riboswitches, ribozymes, and self-splicing introns to be
20
analogous to Cas9 or predictive or its activity in eukaryotes. Ex. 1534, 83-87; Ex. 1535,
21
83-87.
22
23
At page 16, line 3 to page 17, line 18, of Broad Motion 2, it is argued that the Cre, RecA,
EcoRI, and C31 prokaryotic proteins are far less complicated than the Type-II CRISPR-Cas
- 25 -

1
system. At page 15, lines 10-13, of Broad Motion 2, it is argued that the components of a Type-
II CRISPR-Cas must be together in the same place and at the same time. The response is that
prior to December 12, 2012, multicomponent prokaryotic systems including a protein component
and a nucleic acid component had been shown to successfully function in eukaryotic cells. See,
e.g., Ex. 1597 (demonstrating the successful use, in eukaryotic cells, of a heterologous
prokaryotic system containing a protein component and a RNA component); Ex. 1534, 80; Ex.
1535, 80. Further, each of the Cre, RecA, EcoRI, and C31 prokaryotic proteins were shown
to be able to be in the same place, at the same time, as their DNA targets. Ex. 1534, 82; Ex.
1535, 82. Moreover, the prokaryotic RecA protein was known to perform a complicated
10
process that required binding, cleaving, and recombining two separate homologous DNA strands,
11
a three component reaction, in eukaryotic cells. Ex. 1329, at 3094, 3098; Ex. 1534, 82; Ex.
12
1535, 82. Accordingly, examples of Cre, RecA, EcoRI, and C31 and others contradict
13
Broads unsupported argument. Id.
14
For completeness, although not mentioned in Broad Motion 2, it is noted that Dr. Simons
15
cites the protein T7 RNA polymerase as a further example in the same line of argument. Ex.
16
2001, 6.40-6.43 (discussing Wirtz et al. (Ex. 2240)). The response is that Wirtz et al. do not
17
support Broads argument. Wirtz et al. show that the T7 polymerase can access and copy
18
eukaryotic DNA. Ex. 2240, at 4626; MF 111; Ex. 1534, 85-86; Ex. 1535, 85-86.
19
Numerous other prior art references confirm the effectiveness of T7 polymerase in eukaryotic
20
cells. Ex. 1534, 85-86; Ex. 1535, 85-86; see, e.g., Ex. 1579; Ex. 1585; Ex. 1584; Ex.
21
1578. Thus, T7 polymerase would have provided further motivation and an expectation of
22
success to a person of ordinary skill in the art to use the Type-II CRISPR-Cas system in
23
eukaryotic cells. Ex. 1534, 85-86; Ex. 1535, 85-86.
- 26 -

1
Thus, the nonanalogous systems and irrelevant issues cited by Broad would not have
created any uncertainty in the mind of a person of ordinary skill in the art about using the Type-II
CRISPR-Cas in eukaryotic cells. Ex. 1534, 109; Ex. 1535, 109.
4
5
F.
Throughout Broad Motion 2, Broad attempts to make much of selected quotations of UC
inventors. In these statements, the inventors noted that the expectation stated in Jinek 2012 that
the Type-II CRISPR-Cas system would work as a replacement for ZFNs and TALENS in
eukaryotic cells required experimental proof. A first response is that the contemporaneous
Comments of UCs Inventors Do Not Contradict the Reasonable Expectation

of Success and Are Irrelevant
10
statements and actions of UCs inventors demonstrate that prior to December, 2012, they fully
11
expected that the system could be successfully used in eukaryotic cells. Ex. 1534, 30, 35-36,
12
and 68-70; Ex. 1535, 30, 35-36, and 68-70. A second response is that those comments are
13
irrelevant to the question at hand. The question of obviousness in the test for interference-in-fact
14
is determined from the viewpoint of a person of ordinary skill in the art, not an inventor. As
15
clearly shown above, the objective evidence proves that persons of ordinary skill in the art
16
expected that the Type-II CRISPR-Cas system could be used in eukaryotic cells and were
17
actually able to quickly demonstrate it using off-the-shelf reagents and routine methods before
18
and after Broads assumed but unproven prior art cut-off of December 12, 2012.
19
At page 4, lines 4-8, of Broad Motion 2, it is argued that in January of 2013, Dr. Doudna
20
and Dr. Jinek stated that it was not known whether such a bacterial system [the Type-II
21
CRISPR-Cas system] would function in eukaryotic cells. It is further argued that Dr. Doudna
22
stated, in 2014: Our 2012 paper [Jinek 2012] was a big success, but there was a problem. We
23
werent sure if CRISPR/Cas9 would work in eukaryotesplant and animal cells. (citing Ex.
24
2207 at 3). See also Broad Motion 2, at p. 18, ll. 12-19. At page 4, line 14, of Broad Motion 2,
- 27 -

1
it is argued that Dr. Doudnas observations are consistent with the state of the art in 2012.
The response is that a person of ordinary skill in the art would, and in fact did, understand
that these statements do not reflect uncertainty about the widely-held expectation that the system
would work in a eukaryotic cell, but simply reflect that the confirmatory experimental results had
not yet been reported. These statements simply provided an introduction to experiments that
confirmed that the system facilitates site-specific genome targeting in eukaryotic cells as widely
expected. See Ex. 1534, 30, 35-36, and 68-70; Ex. 1535, 30, 35-36, and 68-70; Ex. 1057,
at p. 4 and Fig. 1E; see also Ex. 1555, at p. 61, ll. 11-13; see also Ex. 1471 and Ex. 1152, at p.
1659. Additionally, the disclosure of UCs first provisional patent application, which described
10
and enabled methods of using a Type-II CRISPR-Cas system in eukaryotic cells, demonstrates
11
that UCs inventors expected that Type-II CRISPR system could be used in a eukaryotic cell.
12
MF 112; Ex. 1534, 70; Ex. 1535, 70; see also Ex. 1003, at [00124]-[00129], [00165]-
13
[00177], [00186]-[00188], [00216], [00178]-[00179], and Figs. 1-4.
14
In further response, because the cited comments came after the adaptation to eukaryotic
15
cells had already been accomplished and disclosed, they could not have affected the expectations
16
of a person of ordinary skill in the art in 2012. Moreover, it has long been recognized that
17
[o]bviousness does not require certainty that a modification to the prior art will work, only a
18
reasonable expectation of success is required, which is more than proven by the prior and
19
contemporaneous evidence. See In re Longi, 759 F.2d 887, 897 (Fed. Cir. 1985). Obviousness
20
does not require absolute predictability of success. In re Droge, 695 F.3d 1334, 1338-39 (Fed.
21
Cir. 2012); see also Par Pharm., Inc. v. TWi Pharm., Inc., 773 F.3d 1186, 1198 (Fed. Cir. 2014).
22
Moreover, Broads selective quotations ignore more contemporaneous statements by Dr.
23
Doudna that are inconsistent with Broads argument. For example, in an interview published on
- 28 -

1
June 28, 2012months before Broads first provisional application was filedDr. Doudna
explained UCs recent discovery as follows: Weve discovered the mechanism behind the
RNA-guided cleavage of double-stranded DNA that is central to the bacterial acquired immunity
system. MFs 113-115; Ex. 1546, at 1; Ex. 1534, 35; Ex. 1535, 35. She went on to predict
that Our results could provide genetic engineers with a new and promising alternative to
artificial enzymes for gene targeting and genome editing in bacteria and other cell types. Id.
(emphasis added). The report went on to explain how Dr. Doudna and her colleagues were in the
process of testing whether the system would work in eukaryotic organisms including fungi,
worms, plants and human cells. Id. At the time, Dr. Doudna explained that [a]lthough weve
10
not yet demonstrated genome editing, given the mechanism we describe it is now a very real
11
possibility. Id. These positive, forward-looking, statements were made, and published, prior to
12
Broads first provisional application. Id.
13
At page 8, lines 1-4, of Broad Motion 2, it is argued that Dr. Doudna indicated that she
14
experienced many frustrations trying to get CRISPR to work in human cells and that if the
15
system could be made to work in human cells, it would be a really profound discovery. (citing
16
Ex 2230, at 2). See also Broad Motion 2, at p. 2, ll. 20-21, p. 10, ll. 1-4, p. 13, ll. 11-14. The
17
response is that these citations, which are from articles that published in non-peer-reviewed
18
journals after UC had already successfully applied the Type-II CRISPR system to eukaryotic
19
cells, do not convey that Dr. Doudna expected that the Type-II CRISPR-Cas system would not
20
work in eukaryotes. To the contrary, Dr. Doudna was exploring how well CRISPR/Cas9 would
21
work in eukaryotic cells. Ex. 2207, at p. 3; Ex. 1534, 69; Ex. 1535, 69. The 2013 eLife
22
publication, about seven months after Jinek 2012, is proof that UC was quickly able to apply the
23
Type-II CRISPR-Cas system to eukaryotic cells. Indeed, as Dr. Simons testified during his
- 29 -

1
deposition, [o]ne never does an experiment without the belief that it might work under certain
circumstances. MF 116; Ex. 1555, at p. 178, ll. 10-12; Ex. 1534, 69; Ex. 1535, 69. These
after-the-fact statements in non-technical journals do not, therefore, demonstrate that persons of
ordinary skill in the art lacked a reasonable expectation of success in applying the Type-II
CRISPR system to eukaryotic cells. Indeed, in view of the vast body of knowledge of other
prokaryotic systems that had been successfully applied to eukaryotic cells and the conventional
techniques that were available to do so, a person of ordinary skill in the art would have had a
reasonable expectation that the Type-II CRISPR system of UCs claims could be used in
eukaryotic cells.
10
Further, as acknowledged by Broad, the 2013 eLife paper was published after use of the
11
Type-II CRISPR system in eukaryotes had been demonstrated, and could not have affected the
12
understanding of a person of ordinary skill in the art at the relevant time. And extensive
13
contemporaneous evidence demonstrates that many independent research groups immediately
14
used the Type-II CRISPR system in eukaryotic cells, and were able to quickly do so following
15
the disclosure in Jinek 2012. As shown above, this is a case where the state of the art, which
16
included several analogous examples of success, shows that a skilled artisan would have had a
17
resoundingly reasonable expectation of success in deriving the claimed invention in light of the
18
teachings of the prior art. See In re Kubin, 561 F.3d 1351, 1360 (Fed. Cir. 2009). The cited
19
statements cannot rebut the objective fact that persons of ordinary skill in the art were motivated
20
to move the Type-II CRISPR system into eukaryotic cells, had an expectation of success in doing
21
so, and did so before Broad filed its first patent application.
22
23
At page 9, lines 5-22, of Broad Motion 2, it is argued that Dr. Doudna was interviewed
about the first demonstrations of successful function of Type-II CRISPR-Cas systems in
- 30 -

1
eukaryotic cells and stated that this work would remove a huge bottleneck relating to
techniques for making these modifications in animals and humans.
The response is that in the context of the report of the interview, it is clear that the text is
referring to the fact that the Type-II CRISPR-Cas system as a whole would be important because
it made it much easier to research the effects of modifications in eukaryotic organisms. This
understanding of the quoted text is consistent with Dr. Doudnas quoted remarks that were
published on June 28, 2012. Ex. 1546, at 1. As noted by other independent commentators, it
was UCs discovery that launched a revolution in research, of which Broad was only one of
many groups to build upon. Ex. 2231.
10
11
12
V.
CONCLUSION
For at least the foregoing reasons, Broads Motion 2 should be denied.
Respectfully submitted,
By /Todd R. Walters/
Registration No. 34,040
Alexandria, Virginia 22314
Telephone (703) 836-6620
Facsimile (703) 836-2021
Counsel for UC and Vienna
By: / Sandip H. Patel /

Sandip H. Patel, Esq.
MARSHALL GERSTEIN & BORUN LLP
6300 Willis Tower
233 South Wacker Drive
Telephone (312) 474-6300
Facsimile (312) 474-0448
spatel@marshallip.com
Counsel for EC
Date: August 15, 2016
13
- 31 -

EXHIBIT
DESCRIPTION
NO.
U.S. Patent Application No. 13/842,859, filed on March 15, 2013, to Jennifer Doudna
1001
et al. (the 859 Application)
U.S. Provisional Application No. 61/652,086, filed on May 25, 2012, to Martin Jinek
1003
et al. (the 086 Provisional or the First Provisional)
U.S. Patent No. 8,697,359, issued on April 15, 2014, to Feng Zhang (the 359
1007
Patent)
U.S. Patent No. 8,889,356, issued on November 18, 2014, to Feng Zhang (the 356
1010
Patent)
Dai et al., The Transcription Factors GATA4 and dHAND Physically Interact to
1029
Synergistically Activate Cardiac Gene Expression Through a p300-dependent

Mechanism, 277(27) J. BIOL. CHEM. 24390-24398 (2002) (Dai)
Gustafsson et al., Codon Bias and Heterologous Protein Expression, 22(7) TRENDS
1030
BIOTECHNOL. 346-353 (2004) (Gustafsson)
43 METHODS IN CELL BIOLOGY, Protein Expression In Animal Cells, Chapters 2, 3, 6,
1031
9 (Michael G. Roth ed., 1994) (Roth)
Boden et al., Efficient Gene Transfer of HIV-1-Specific Short Hairpin RNA into
1039
Human Lymphocytic Cells Using Recombinant Adeno-associated Virus Vectors, 9(3)

MOL. THER. 396-402 (2004) (Boden)
MOLECULAR CLONING: A LABORATORY MANUAL, Chpt. 16 (J. Sambrook & D.
1040
Russell, 3rd ed. 2001) (Sambrook)
1-1

EXHIBIT
DESCRIPTION
NO.
Cong et al., Multiplex Genome Engineering Using CRISPR/Cas Systems, 339(6121)
1055
SCIENCE 819-823 (2013) with Supplemental Material (Cong)
Mali et al., RNA-Guided Human Genome Engineering via Cas9, 339(6121) SCIENCE
1056
823-826 (2013) with Supplemental Materials (Mali)
Jinek et al., RNA-Programmed Genome Editing in Human Cells, 2 ELIFE e00471
1057
(2013) (Jinek 2013)
Hwang et al., Efficient In Vivo Genome Editing Using RNA-Guided Nucleases, 31(3)
1058
NAT. BIOTECHNOL. 227-229 (2013) (Hwang)
Cho et al., Targeted Genome Engineering in Human Cells With the Cas9 RNA1059
Guided Endonuclease, 31(3) NAT. BIOTECHNOL. 230-232 (2013) with Supplemental

Information (Cho)
Shen et al., Generation of gene-modified mice via Cas9/RNA-mediated gene
1060
targeting, 23(5) CELL RES. 720-723 (2013) (Shen)
Carroll, A CRISPR approach to gene targeting, 20(9) MOLECULAR THERAPY 1658-60
1152
(2012)
Jinek et al., A programmable dual-RNA-guided DNA endonuclease in adaptive
1155
bacterial immunity, 337(6096) SCIENCE 816-821 (2012) (Jinek 2012)
U.S. Patent Publication No. 2010/0076057, published on March 25, 2010 to
1161
Sontheimer et al.
Anderson et al., A simple method for the rapid generation of recombinant adenovirus
1194
vectors, 7 GENE THERAPY 1034-1038 (2000)
1-2

EXHIBIT
DESCRIPTION
NO.
Behr et al., Efficient gene transfer into mammalian primary endocrine cells with
1200
lipopolyamine-coated DNA, 86 PNAS 6982-6986 (1989)
Bergemann et al., Excision of specific DNA-sequences from integrated retroviral
1203
vectors via site-specific recombination, 23(21) NUCL. ACIDS RES. 4451-4456 (1995)
Choulika, et al., Transfer of single gene-containing long terminal repeats into the
1218
genome of mammalian cells by a retroviral vector carrying the cre gene and the loxP
site, 70(3) J. VIROL. 1792-1798 (1996)
Dykxhoorn et al., Killing the Messenger: Short RNAs That Silence Gene Expression,
1229
4 NATURE REV. 457-467 (2003)
Fechheimer et al., Transfection of mammalian cells with plasmid DNA by scrape
1233
loading and sonication loading, 84 PNAS 8463-8467 (1987)
Fieck et al., Modifications of the E.coli Lac repressor for expression in eukaryotic
1235
cells: effects of nuclear signal sequences on protein activity and nuclear

accumulation, 20(7) NUCL. ACIDS RES. 1785-1791 (1992)
Fischer-Fantuzzi and Vesco, Cell-Dependent Efficiency of Reiterated Nuclear Signals
1236
in a Mutant Simian Virus 40 Oncoprotein Targeted to the Nucleus, 8(12) MOL. CELL
BIOL. 5495-5503 (1988)
Gorman et al., High efficiency gene transfer into mammalian cells, B307 PHIL.
1248
TRANS. R. SEC. LAND. 343-346 (1984)
1-3

EXHIBIT
DESCRIPTION
NO.
Hashimoto et al., A novel method for transformation of intact yeast cells by
1260
electroinjection of plasmid DNA, 21 APPLIED MICROBIOL. BIOTECHNOL. 336-339

(1985)
Lambowitz and Zimmerly, Group II introns: mobile ribozymes that invade DNA, 3(8)
1276
COLD SPRING HARB PERSPECT BIOL. a003616 (2011)
Li et al., In vivo genome editing restores haemostasis in a mouse model of
1283
haemophilia, 475 NATURE 217-223 (2011)
Lombardo et al., Gene editing in human stem cells using zinc finger nucleases and
1285
integrase-defective lentiviral vector delivery, 25(11) NAT. BIOTECHNOL. 1298-1306

(2007)
Mastroianni et al., Group II intron-based gene targeting reactions in eukaryotes, 3(9)
1293
PLOS ONE. e3121 (2008)
Morgan et al., Inducible Expression and Cytogenetic Effects of the EcoRI Restriction
1302
Endonuclease in Chinese Hamster Ovary Cells, 8(10) MOL. CELL. BIOL. 4204-4211
(1988)
Neumann et al., Gene transfer into mouse lyoma cells by electroporation in high
1307
electric fields, 1(7) EMBO JOURNAL 841-845 (1982)
OHare et al., Transformation of mouse fibroblasts to methotrexate resistance by a
1310
recombinant plasmid expressing a prokaryotic dihydrofolate reductase, 78(3) PNAS

1527-1531 (1981)
1-4

EXHIBIT
DESCRIPTION
NO.
Patterson et al., Codon optimization of bacterial luciferase (lux) for expression in
1315
mammalian cells, 32(3) J. IND. MICROBIOL. BIOTECHNOL. 115-123 (2005)
Planey et al., Inhibition of Glucocorticoid-induced Apoptosis in 697 Pre-B
1319
Lymphocytes by the Mineralocorticoid Receptor N-terminal Domain, 277(44) J.

BIOL. CHEM. 42188-42196 (2002)
Raymond and Soriano, High-efficiency FLP and C31 site-specific recombination in
1327
mammalian cells, 2(1) PLOS ONE. e162 (2007)
Reiss et al., RecA protein stimulates homologous recombination in plants, 93 PROC.
1329
NATL. ACAD. SCI. USA 3094-3098 (1996)
Sandy et al., Mammalian RNAi: a practical guide, 39 BIOTECHNIOUES 215-224
1332
(2005)
Sauer, Functional expression of the cre-lox site-specific recombination system in the
1335
yeast saccharomyces cerevisiae, 7(6) MOL. CELL. BIOL. 2087-2096 (1987) (Sauer
1987)
Sauer and Henderson, Site-specific DNA recombination in mammalian cells by the
1336
Cre recombinase of bacteriophage P1, 85 PROC. NATL. ACAD. SCI. USA 5166-5170
(1988)
Schramm and Hernandez, Recruitment of RNA polymerase III to its target promoters,
1337
16(20) GENES DEV. 2593-2620 (2002)
DiCarlo et al., Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas
1372
systems, 41(7) NUCL. ACIDS RES. 4336-4343 (2013) (DiCarlo)
1-5

EXHIBIT
DESCRIPTION
NO.
1471
Brouns, A Swiss Army Knife of Immunity, 337 SCIENCE 808-809 (2012)

Golic, RNA-Guided Nucleases: A New Era for Engineering the Genomes of Model
1473
and Nonmodel Organisms, 195 GENETICS 303-308 (2013)
REDACTED Email from Shuailiang Lin to Jennifer Doudna, dated February 28,
1475
2015, with attachments
Suggestion of Interference, filed April 13, 2015, in U.S. Patent Application No.
1529
13/842,859 without Appendices
1534
Second Declaration of Carol Greider, Ph.D.
1535
Second Declaration of Dana Carroll, Ph.D.

U.S. Provisional Application No. 61/717,324, filed October 23, 2012 to Seung Woo
1545
Kim et al. (Toolgen Provisional)
Yarris, Programmable DNA Scissors Found for Bacterial Immune System (June 28,
1546
2012) downloaded from http://newscenter.lbl.gov/2012/06/28/programmable-dnascissors/ on June 13, 2016

Hwang et al., Efficient genome editing in zebrafish using a CRISPR-Cas system,
1554
31(3) NATURE BIOTECHNOLOGY 227-229 (2013) with Supplementary Materials
1555
Simons Deposition Transcript, July 18, 2016 with errata
1556
Simons Deposition Transcript, July 19, 2016 with errata
1557
[REDACTED] Email from Jin-Soo Kim to Jennifer Doudna, dated July 16, 2012
[REDACTED] Email from Jin-Soo Kim to Jennifer Doudna, dated October 3, 2012
1558
with attachment
1-6

EXHIBIT
DESCRIPTION
NO.
1559
Email from George Church to Jennifer Doudna, dated November 14, 2012
[REDACTED] Email from George Church to Jennifer Doudna, dated December 8,
1560
2012 with attachments
Ran, F. A. CRISPR/Cas9: Tools and Applications for Eukaryotic Genome Editing, 26
1561
NORTH AMERICAN AGRICULTURAL BIOTECHNOLOGY COUNCIL REPORT 69-81 (2014)
1571
Declaration of Bernardette Rossi

Lanza et al., Evaluating the influence of selection markers on obtaining selected
1574
pools and stable cell lines in human cells, 8 J. BIOTECHNOL. 811-821 (2013)
Mullen et al., Transfer of the bacterial gene for cytosine deaminase to mammalian
1575
cells confers lethal sensitivity to 5-fluorocytosine: A negative selection system, 89

PNAS 33-37 (1992)
Ill and Chiou, Gene Therapy Progress and Prospects: Recent progress in transgene
1576
and RNAi expression cassettes, 12 GENE THERAPY 795-802 (2005)
Brisson et al., A novel T7 RNA polymerase autogene for efficient cytoplasmic
1578
expression of target genes, 6 GENE THERAPY 263-270 (1999)
Fuerst et al., Use of a Hybrid Vaccinia Virus-T7 RNA Polymerase System for
1579
Expression of Target Genes, 7(7) MOL. AND CELL. BIOL. 2538-2544 (1987)
Dulon and Ryan, The bacterial Neo gene confers neomycin resistance to mammalian
1580
cochlear hair cells, 10 NEUROREPORT 1189-1193 (1999)
Mulligan and Berg, Expression of Bacterial Gene in Mammalian Cells, 209 SCIENCE
1581
1422-1427 (1980)
1-7

EXHIBIT
DESCRIPTION
NO.
Truong et al., Retrohoming of a Mobile Group II Intron in Human Cells Suggests
1582
How Eukaryotes Limit Group II Intron Proliferation, 11(8) PLOS GENET. e1005422
(2015)
Serganov and Patel, Ribozymes, riboswitches and beyond: regulation of gene
1583
expression without proteins, 8 NATURE REVIEWS 776-790 (2007)
Lieber et al., Stable High-Level Gene Expression in Mammalian Cells by T7 Phage
1584
RNA Polymerase, 217 METHODS IN ENZYMOLOGY 47-66 (1993)
Lieber et al., High level gene expression in mammalian cells by a nuclear T7-phage
1585
RNA polymerase, 17(21) NUCL. ACIDS RES. 8485-8493 (1989)
Maury et al., Technical advances to genetically engineering human embryonic stem
1594
cells, 3 INTEGR. BIOL. 717-723 (2011)
Nagy et al., Creation and Use of a Cre Recombinase Transgenic Database, Gene
1595
Knockout Protocols, Chpt. 19, pp. 365-378 (Ralf Khn, Wolfgang Worst eds. 2nd ed.
2009)
Karow et al., Site-specific recombinase strategy to create iPS cells efficiently with
1596
plasmid DNA, 29(11) STEM CELLS 1696-1704 (2011)
Chen et al., A Facile System for encoding Unnatural Amino Acids in Mammalian
1597
Cells, 48(22) ANGEW CHEM. INT. ED. ENGL. 4052-4055 (2009)
[REDACTED] Email from Jin-Soo Kim to Emmanuelle Charpentier, Ines Fonfara,
1598
and Jennifer Dounda, dated October 4, 2012
1-8

EXHIBIT
DESCRIPTION
NO.
U.S. Patent Application Publication No. 2015/0322457, published on November 12,
1599
2015 to Kim et al. (the published Kim application)
Office Action dated December 5, 2013 in U.S. Patent Application No. 14/054,414
1601
with Notice of References Cited
Petition and Request Under 37 C.F.R. 1.48(d) to Correct Inventorship, filed on
1602
December 11, 2013 in U.S. Provisional Application No. 61/736,527
2001
Declaration of Dr. Paul Simons, executed May 23, 2016.
2101
U.S. Provisional Patent Application No. 61/736,527, filed December 12, 2012.
2104
Reiss et al., U.S. Patent 6,583,336 B1, June 4, 2003

College of Chemistry, University of California, Berkeley, 9 Catalyst 1-32
2207
(Spring/Summer 2014)
Grens et al., Enzyme Improves CRISPR A smaller Cas9 protein enables in vivo
2213
genome engineering via viral vectors, The Scientist (April 1, 2015), http://www.thescientist.com//?articles.view/articleNo/42580/title/Enzyme-Improves-CRISPR/
Koseki et al., Factors governing the activity in vivo of ribozymes transcribed by RNA
2221
polymerase III, 73 J. Virology 1868-1877 (1999)
Link et al., Engineering ligand-responsive gene-control elements: lessons learned
2223
from natural riboswitches, 16 Gene Therapy 1189-1201 (2009)
Pandika, Rising Stars: Jennifer Doudna, CRISPR Code Killer, OZY (Jan. 7, 2014),
2230
http://www.ozy.com/rising-stars/jennifer-doudna-crispr-code-killer/4690
2231
Pennisi, The CRISPR Craze, 341 Science 833-836 (2013).
1-9

EXHIBIT
DESCRIPTION
NO.
Wirtz et al., Regulated processive transcription of chromatin by T7 RNA polymerase
2240
in Trypanosoma brucei, 26 Nucleic Acids Res. 4626-4634 (1998).
Mali et al., RNA-Guided Human Genome Engineering via Cas9, 339 Science 8232258
826 (2013)
Mastroianni et al., Group II Intron-Based Gene Targeting Reactions in Eukaryotes,
2261
PLoS ONE 3(9):e3121 (2008)
Google Trends,
2403
https://www.google.com/trends/explore#q=CRISPR%2C%20Cas9&cmpt=
q&tz= Etc%2FGMT%2B4
1-10

APPENDIX 2 - STATEMENT OF MATERIAL FACTS
Junior Party Alleged Facts 1-54
1.
Clustered Regularly Interspaced Short Palindromic Repeats or CRISPR refers
to several different microbial systems that were discovered to be involved in a bacterial immune
response to invading phages and plasmids. Ex. 2001, Simons 2.1. Response: Admitted.
2.
All of Broads involved claims recite language that explicitly requires operability
of the CRISPR-Cas9 system in a eukaryotic cell. Ex. 2001, Simons 6.1; Paper 43, Broad Clean
Claims. Response: Admitted.
3.
Claim 1 of the Broad 359 patent is directed to A method of altering expression
of at least one gene product comprising introducing into a eukaryotic cell containing and
expressing a DNA molecule having a target sequence and encoding the gene product an
engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats
(CRISPR)--CRISPR associated (Cas) (CRISPR-Cas) system comprising one or more vectors
comprising: a) a first regulatory element operable in a eukaryotic cell operably linked to at least
one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with the
target sequence, and b) a second regulatory element operable in a eukaryotic cell operably linked
to a nucleotide sequence encoding a Type-II Cas9 protein, wherein components (a) and (b) are
located on same or different vectors of the system, whereby the guide RNA targets the target
sequence and the Cas9 protein cleaves the DNA molecule, whereby expression of the at least one
gene product is altered; and, wherein the Cas9 protein and the guide RNA do not naturally occur
together. Paper 43, Broad Clean Claims at p. 3, ll. 215. Response: Admitted.
4.
UCs involved claims do not recite or require that the CRISPR-Cas 9 system
operate within a eukaryotic cell. Ex. 2001, Simons 6.1; Paper 12, Senior Partys Clean Claims.
Response: Admitted.
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5.
UCs involved claims are directed to contacting certain components of a
CRISPR-Cas9 system and a target DNA molecule. Id. Response: Denied.

6.
UC admits, none of [its] claims require performance of the claimed method or
application of the claimed system in a eukaryotic cell. Paper 27, UC Proposed Motions List at p.
7, line 25-p. 8, line 1. Response: Admitted.
7.
UCs involved claims, if treated as prior art, do not anticipate Broads involved
claims. Ex 2001, Simons 6.16.2. Response: Admitted.

8.
Application 61/736,527, Exhibit 2101, Zhang B1, was filed on December 12,
2012. Ex. 1007, B1 (Cover Page Certificate). Response: Admitted.

9.
During the relevant time period, a person of ordinary skill in the art would have a
broad background that includes a strong understanding of the molecular biology and biochemistry
techniques needed to clone, express, isolate, purify, and manipulate proteins and nucleic acids in
the context of both in vitro and in vivo experiments in both prokaryotes and eukaryotes; a Ph.D.
degree in a life sciences discipline, e. g., chemistry, biochemistry, neurobiology; and at least one
year of relevant post-doctoral experience. Ex. 2001, Simons 4.1. Response: Denied.
10.
Prokaryotic proteins, like Cas9, have evolved in the context of prokaryotic cells.
Ex. 2001, Simons 6.33. Response: Insufficient information, therefore unable to admit or
deny.
11.
There is no corollary to the natural, bacterial Cas9 or the CRISPR-Cas9 system in
eukaryotic cells. Ex. 2001, Simons 6.13. Response: Insufficient information, therefore
unable to admit or deny.
12.
The folding of a Cas9 protein in a eukaryotic cell is unpredictable. Ex. 2001,
Simons 6.66.9. Response: Denied.
2-2

13.
The unpredictability is due at least to the different protein folding environment in
a eukaryotic cell compared to a prokaryotic cell. Ex. 2001, Simons 6.9, 6.13. Response:
Denied.
14.
As of December 2012, a person of ordinary skill in this art would have considered
the use of a CRISPR-Cas9 system in eukaryotic systems to be unpredictable at least because of

the many differences between the cellular environment of a prokaryotic or bacterial cell and the
cellular environment of a eukaryotic cell. Ex. 2001, Simons 6.14-6.47. Response: Denied.
15.
Differences between prokaryotic systems and eukaryotic systems include gene
expression, cellular compartmentalization, cellular nucleases, intracellular ion concentrations,

intracellular pH, different types of molecules present in eukaryotic cells that are not native to
bacterial cells, and different interactions of the CRISPR-Cas9 complex and components thereof
with proteins or nucleic acids that are present in eukaryotic cells and not native to bacterial cells.
Ex. 2001, Simons 6.28. Response: Denied.
16.
Engineering a delivery system for proper expression or presence of the CRISPR-
Cas9 system would involve temporal and spatial requirements, i.e., the components must be
together at the same time and in the same place as each other and the target for the system to
work successfully. Ex. 2001, Simons 6.32. Response: Insufficient information, therefore
unable to admit or deny.
17.
In a eukaryotic cell, translation of the Cas9 protein takes place in the cytoplasm,
whereas transcription of the RNA component of the CRISPR-Cas9 complex takes place in the
nucleus. Ex. 2001, Simons 6.68. Response: Denied.
18.
As of 2012, one of ordinary skill in the art could not have predicted whether
intracellular degradation pathways, for both protein and / or RNA components would degrade the
2-3

molecules or otherwise inhibit complexing of the components. Ex. 2001, Simons 6.30.
Response: Denied.
19.
Eukaryotic chromosomes are composed of chromatin. Ex. 2001, Simons 6.29.
Response: Denied.
20.
Chromatin is a complex and tightly-packed structure composed of genomic DNA
complexed with proteins (primarily histones). Ex. 2001, Simons 6.29. Response: Denied.
21.
Prokaryotic cells do not have a nucleus and generally have a single chromosome
that is not complexed with histones to form chromatin. Ex. 2001, Simons 6.29. Response:
Denied.
22.
A skilled person in this art on December 12, 2012, would have recognized that
components unique to bacterial cells may not function in a eukaryotic cell and could be
deleterious to a eukaryotic cell. Ex. 2001, Simons 6.35. Response: Denied.
23.
The known disclosures as of December 12, 2012, did not provide sufficient
information for one of skill in this art to engineer a CRISPR-Cas9 system for use in eukaryotic
cells with any reasonable expectation of success. Ex. 2001, Simons 2.11. Response: Denied.
24.
The 2012 published experiments of UCs inventors, Doudna et al, as set forth in
the Jinek 2012 reference and included in the priority applications of UCs 859 application filed
in 2012, only contacted isolated components of a CRISPR-Cas9 system with a naked DNA target
in a cell-free environment in in vitro experiments. Ex. 2001, Simons 6.1-6.4, 6.29. Response:
Denied.
25.
Even after UCs in vitro experiments in 2012, Dr. Doudna experienced many
frustrations getting CRISPR to work in human cells and believed that development of a CRISPR
2-4

system for use in eukaryotic cells would be a profound discovery Ex. 2230, Pandika at 3; Ex.
2001, Simons 6.50. Response: Denied.
26.
Dr. Dana Carrolls contemporaneous expression of doubt about the operability of
the CRISPR-Cas9 system in eukaryotic cells from the experiments of Jinek 2012 (Ex. 1152,
Carroll 2012) recognizes that one of skill in this art could not have had any reasonable
expectation of success in adapting CRISPR-Cas9 to function eukaryotic cells. Ex. 1152, Carroll
2012 at 1660; Ex. 2001, Simons 6.4. Response: Denied.
27.
Neither UC nor Dr. Carroll points to any methods and materials that would have
made it routine to apply a CRISPR-Cas system in a eukaryotic cell. Ex. 2001, Simons 6.576.72. Response: Denied.
28.
In the suggestion of interference (Ex. 1529, Suggestion filed April 13, 2015 at 27,
ll. 22-23), the arguments and examples relied upon by UC fail to support UCs conclusion that
introduction of the Type-II CRISPR-Cas System in eukaryotic cells would have been routine or
that one of ordinary skill in the art would have had a reasonable expectation of success. Ex.
2001, Simons 6.57. Response: Denied.
29.
The Cre, RecA, EcoRI, C31, TALEN, and ZFN systems cited by UC in its
suggestion of interference are far less complicated than the Type-II CRISPR-Cas system at issue
in the instant matter; in each example cited by UC, the proteins are not required to form a
complex with a DNA-targeting RNA to function successfully. Ex. 2001, Simons 6.67.
Response: Denied.
30.
None of the prior systems cited by Dr. Carroll or relied upon by UC in its
suggestion of interference involve a bacterial protein that is RNA-guided and hence involved
protein and RNA components. Ex. 2001, Simons 6.68. Response: Denied.
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31.
The bacterial protein and the RNA can present issues of cellular toxicity in the
eukaryotic environment, and when expressed in vivo in the eukaryotic cell, the protein is
expressed in the cytoplasm and the RNA is in the nucleusaspects of the bacterial CRISPRCas9 system that made modifying it for functioning in the environment of a eukaryotic cell
wholly unpredictable at the December 12, 2012 filing date of Zhang B1, Ex. 2101. Ex. 2001,
Simons 6.68. Response: Denied.
32.
Ex. 1335, Sauer 1987 and Ex. 1336, Sauer 1988 teach that prokaryotic systems
cannot be presumed to be able to be effectively transferred to eukaryotes, stating that the ability
of the Cre protein to access a lox site placed on a chromosome and then to perform site specific
synapsis of DNA and reciprocal recombination may be highly dependent on surrounding
chromatin structure and on the particular location within the genome of the lox site; some regions
of the genome may be inaccessible to a bacterial recombinase, for example. Ex. 1336, Sauer
1988 at 5170. Response: Denied.
33.
It was known in the prior art as of 2012, that RNA-based, self-splicing Group II
introns are found in prokaryotic organisms and even in the mitochondria and chloroplasts of
lower eukaryotes, but that obstacles include nuclear accessibility of RNPs and suboptimal Mg2+
concentrations. Ex. 2001, Simons 6.376.38; Ex. 1276, Lambowitz et al. 2011 at 14; Romani
et al., (2002); Ex. 2261, Mastroianni et al., (2008). Response: Insufficient information,
therefore unable to admit or deny.
34.
While the authors of the Reiss et al. publication (Ex. 1329, Reiss) were able to
successfully use a prokaryotic protein (RecA) in eukaryotic cells (tobacco plant cells) (Ex. 1529,
Suggestion filed April 13, 2015 at 28, ll. 17-22), their success was unpredictable and the authors
2-6

were awarded a patent for their accomplishment (US Patent No. 6,583,336) (Ex. 2104).
Response: Denied.
35.
A person of skill in the art would understand that RecA has a corresponding
homologous protein present in eukaryotic cells, while Cas9 has no equivalent in a eukaryotic cell
and, the work of Reiss et al. with RecA would not provide any basis for a reasonable expectation
of success with respect to adaptation a CRISPR-Cas9 system for eukaryotes. Ex. 2001, Simons
6.65. Response: Denied.
36.
The success with ZFNs and TALENs in eukaryotes would not provide any basis
for a reasonable expectation of success with respect to adaptation of a CRISPR-Cas9 system for
eukaryotes because, unlike CRISPR-Cas9, ZFNs and TALENs are not purely prokaryotic in
nature (the eukaryote-derived portion of ZFNs comprise DNA-binding domains critical to the
proteins function in a eukaryote. while the equivalently critical DNA binding domains of
TALENs originate in bacteria, the TALE proteins from which they are derived have their
function in plants, i.e. eukaryotes). Ex. 2001, Simons 6.696.70. Response: Denied.
37.
ZFN and TALEN systems are very different from CRISPR-Cas, which includes an
RNA-guided protein that had never been previously known to express or function in a eukaryotic
cell . Ex. 2001, Simons 6.67-6.70. Response: Denied.
38.
As Dr. Carroll noted in 2012 that zinc fingers and TALE modules come from
natural transcription factors that bind their targets in a chromatin context. This is not true of the
CRISPR components. Ex. 1152, Carroll 2012 at 1660. Response: Insufficient information,
therefore unable to admit or deny.
39.
Prior to the Broads publication of the work reflected in its first two provisional
applications in Ex. 1055, Cong, no group had shown that the RNA constructs and Cas9 protein of
2-7

the CRISPR-Cas system could function in a eukaryotic cell. Ex. 2001, Simons 2.10.
Response: Denied.
40.
Dr. Feng Zhang and his colleagues invented engineered CRISPR-Cas9 systems
that function in eukaryotic cells. Ex. 2001, Simons 2.13. Response: Denied.
41.
The invention of CRISPR-Cas systems for eukaryotic cells, as in Ex. 2101, Zhang
B1; Ex. 1055, Cong, and the eukaryotic subject matter claims of the Broad patents was
recognized as a pioneering. Ex. 2001, Simons 2.13-2.15. Response: Denied.
42.
Google provides real-time data on internet searches, known as Google Trends, and
that such a search as to CRISPR or Cas9 is available from the following link:
https://www.google.com/trends/explore#q=CRISPR%2C%20Cas9&cmpt=q&tz=Etc%2FGMT%
2B4. Ex. 2403. Response: Insufficient information, therefore unable to admit or deny.
43.
This real-time data, as shown by the following Google Trends graph understood to
reflect inclusion of search terms CRISPR or Cas9 over time, demonstrates that January 2013
coinciding with the publications on work in eukaryotic cells, (Exs. 1055 and 2258)appears as
the beginning of significant interest in the CRISPR field. Ex. 2403.
Response: Denied.
44.
As of May 2016, the Zhang laboratory, including through Addgene, has
distributed more than 30,000 CRISPR-Cas9 reagents, stemming from the eukaryotic work of the
2-8

Zhang lab, and Cong et al. (Ex. 1055) has more than 1650 acknowledging citations to date and is
the most highly cited CRISPR publication. Ex. 2001, Simons 2.14. Response: Insufficient
information, therefore unable to admit or deny.
45.
In 1999, Koseki et al. reported that the efficacy of RNA ribozymes molecules in
vitro is not predictive of functional activity in vivo, for a variety of reasons, including degradation
of the ribozyme in the eukaryotic cell and inability to colocalize with its target in the eukaryotic
cell. Ex. 2221, Koseki at 187576. Response: Denied.
46.
Despite great interest in the use of riboswitches as a means of controlling gene
expression in other contexts, the creation of riboswitches that function as desired in mammalian
systems has proved intractable to date. Ex. 2223, Link. Response: Denied.
47.
Link et al. (Ex. 2223, Link at 1192) note, a few TPP riboswitches are the only
validated metabolite-binding riboswitches in eukaryotes and many of the engineered

riboswitches that function well in vitro fail to provide useful function when translated to
eukaryotic cells. Ex. 2001, Simons 6.47. Response: Denied.
48.
It was uncertain whether a Cas protein could access target DNA in a eukaryotic
cell. Ex. 2001, Simons 6.29. Response: Insufficient information, therefore unable to
admit or deny.
49.
Different cellular conditions can impact protein folding, which is of particular
importance given that CRISPR-Cas9 systems must undergo significant conformational changes
both when binding the guide RNA, and when binding and cleaving the target DNA. Ex. 2001,
Simons 6.13, 6.33. Response: Denied.
2-9

50.
A person of ordinary skill in the art during the relevant time period could not have
predicted whether eukaryotic enzymes would cleave the RNA molecules critical for the
functioning of the CRISPR-Cas9 system. Ex. 2001, Simons 6.15. Response: Denied.
51.
The CRISPR-Cas9 systems requirement for magnesium would have suggested to
a skilled person that the prokaryotic environment, which includes a high concentration of
magnesium, might be essential for the operation of CRISPR systems in vivo. Ex. 2001, Simons
6.13, 6.38-6.39. Response: Denied.
52.
The contemporaneous evidence confirms that, given the nature of the bacterial-
derived CRISPR system and the substantial differences between the prokaryotic and eukaryotic
environments, skilled artisans would not have reasonably predicted that CRISPR systems would
function in eukaryotes, even after successful in vitro DNA cleavage experiments such as reported
in Jinek 2012. Ex. 2001, Simons 6.48-6.51. Response: Denied.
53.
Dr. Doudna stated that [w]e werent sure if CRISPR/Cas9 would work in
eukaryotesplant and animal cells. Ex. 2207 at 3. Response: Admitted.

54.
Another scientist in the field noted, [i]ts not trivial to make CRISPR/Cas
systems work in eukaryotic cells One thing is to have them in silico and have a sequence
and another thing is to do the experiments and make it work. Ex. 2213, Grens at 2 (quoting
Luciano Marraffini). Response: Insufficient information, therefore unable to admit or deny.
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Senior Party Facts in Support of Opposition 2
55.
The eukaryotic cell limitation is the only difference between the claims of the
Parties that is substantively addressed in Broad Motion 2. Ex. 1534, 19; Ex. 1535, 19.
56.
Dr. Simons acknowledged that in 2012 there would have been motivation to use
the Type-II CRISPR-Cas system in a eukaryotic cell. Ex. 1555, at p. 100, l. 17 - p. 102, l. 6.
57.
U.S. Patent Application Publication No. 20150322457 (Ex. 1599) (the Kim
Application) discloses and claims a Type-II CRISPR-Cas composition for cleaving a target
nucleic acid sequence in a eukaryotic cell. Ex. 1599, [0013], Claim 58.
The Kim Provisional cites UCs Jinek et al., SCIENCE 2012; 337:816821 (Jinek
58.
2012) (Ex. 1155), stating:

Recently, Jinek et al. (2) elegantly demonstrated that a single-chain chimeric
RNA produced by fusing an essential portion of crRNA and tracrRNA could
replace the two RNAs in the Cas9 /RNA complex to form a functional
endonuclease, raising the possibility of using this system for genome editing in
cells and organisms.
Ex. 1545, at 7 (p. 1 of the specification).
59.
U.S. Patent Application No. 61/717,324 (Ex. 1545) (the Kim Provisional), filed
October 23, 2012, describes an experiment that is summarized as follows:

We co-transfected the Cas9-encoding plasmid, the guide RNA, and the RFP -GFP
reporter plasmid into human embryonic kidney (HEK) 293T cells, and found that
GFP- expressing cells were obtained only when the cells were co-transfected with
the Cas9 plasmid and the guide RNA (Fig. 2), demonstrating that [RNA -guided
endonucleases] could recognize and cleave the target DNA sequence in cultured
human cells.
Ex. 1545, at 9 (p. 3 of the specification).
60.
The Kim Provisional and the Kim Application each disclosed the use of a Type-II
CRISPR-Cas system in eukaryotic cells prior to the filing of Broads first application. Ex. 1534,
23-25; Ex. 1535, 23-25.
2-11

61.
UCs 859 Application (Ex. 1001) with an effective date of UCs first provisional
application, U.S. Patent Application No. 61/652,086, filed May 25, 2012 (Ex. 1003) is prior art
to Broad under 35 U.S.C. 102(e).
62.
UCs first provisional application disclosed expression of a Type-II CRISPR-Cas
system in eukaryotic cells (Ex. 1003, 124-129; Ex. 1001; 25, 103-105; 119-120; 244-251;
277-282), or supplying a Cas9 site-directed modifying polypeptide to eukaryotic cells as RNA or
protein (Ex. 1003, 177-179; Ex. 1001; 287-289), for purposes of targeting DNA contained
therein (Ex. 1003, 165-177; 186-188, 216; Ex. 1001; 274-275). See also Ex. 1003; Figs.
1-4; Ex. 1001; Figs. 1-3, 9.
63.
U.S. Patent Publication No. 2010/0076057, filed September 23, 2009 by
Sontheimer et al. (Ex. 1161) (Sontheimer) suggested that CRISPR systems could be used in
eukaryotic cells. Ex. 1534, 22; Ex. 1535, 22; Ex. 1161, at 0007.
64.
Sontheimer disclosed conventional techniques available to adapt CRISPR systems
to eukaryotic cells, including expression vectors, nuclear localization signals, and codon
optimization. Ex. 1534, 22; Ex. 1535, 22; Ex. 1161, at 0009, 0042, 0054, 0058, 0060.
65.
Jinek 2012 disclosed the potential to exploit the [Type-II CRISPR-Cas] system
for RNA-programmable genome editing months before Broad filed its first provisional
application. Ex. 1155, Abstract; Ex. 1534, 30; Ex. 1535, 30.
66.
Jinek 2012 disclosed the exciting possibility of developing a simple and versatile
RNA-directed system to generate dsDNA breaks for genome targeting and editing. Ex. 1155,
at 816, col. 2-3; Ex. 1534, 30; Ex. 1535, 30.
67.
Jinek 2012 stated:

Zinc-finger nucleases and transcription-activatorlike effector nucleases have
attracted considerable interest as artificial enzymes engineered to manipulate
2-12

genomes (3538).We propose an alternative methodology based on RNAprogrammed Cas9 that could offer considerable potential for gene-targeting and
genome-editing applications.
Ex. 1155, at 820, col. 3.
68.
Zinc-finger nucleases (ZFNs) and transcription-activatorlike effector nucleases
(TALENs) were, at the time of Jinek 2012, the state of the art for DNA cleavage and editing in
eukaryotic cells. Ex. 1534, 30; Ex. 1535, 30.
69.
A person of ordinary skill in the art would have understood the proposal in Jinek
2012 to replace ZFNs and TALENS with the programmable Type-II CRISPR-Cas system to be a
proposal to use the Type-II CRISPR-Cas system for eukaryotic gene editing. Ex. 1534, 31;
Ex. 1535, 31.
70.
In commentary that accompanied the publication of Jinek 2012, Stan Brouns of
Wageningen University wrote

Jinek et al. realized that a highly specific, customizable RNA-directed DNA
nuclease could be useful to edit whole genomes. Based on the 20-nucleotide guide
section of the crRNA, the enzyme could theoretically introduce breaks at unique
sites in any eukaryotic genome. . . . Introducing DNA breaks at desired loci using
just Cas9 and a chimeric crRNA would be a substantial improvement over
existing gene-targeting technologies, such as zinc finger nucleases and
transcription activatorlike effector nucleases, as these require protein
engineering for every new target locus (10). Efficient gene repair strategies in
cells from patients, and the reintroduction of repaired cells, could become
increasingly important for treating many genetic disorders.
Ex. 1471; Ex. 1534, 32; Ex. 1535, 32.
71.
Before December 12, 2012, at least two independent groups had submitted
manuscripts for publication that demonstrated use of the Type-II CRISPR-Cas system in
eukaryotic cells. Ex. 1534, 63; Ex. 1535, 63.
72.
Manuscripts by Mali et al. (Ex. 1056), Cho et al, (Ex. 1059), Jinek et al. (Ex.
1057), and Hwang et al. (Exs. 1058, 1554) were submitted between October 2012 and December
2-13

18, 2012 and demonstrated use of the Type-II CRISPR-Cas system in eukaryotic cells. Ex.
1534, 63-68; Ex. 1535, 63-68.
73.
Mali et al., Cho et al., Jinek et al., and Hwang et al. were published in January
2013. Ex. 1534, 63-68; Ex. 1535, 63-68.

74.
Mali et al., Cho et al, Jinek et al., and Hwang et al. specifically cited Jinek 2012
as a basis of their work. Ex. 1534, 72; Ex. 1535, 72.

75.
Each of Mali et al., Cho et al, , Jinek et al., and Hwang et al. was submitted by a
different independent research group. Ex. 1534, 63-68 and 72; Ex. 1535, 63-68 and 72.
76.
George Church and Jin Soo Kim, lead investigators on the Mali and Cho papers,
respectively, each independently contacted Drs. Doudna and Charpentier prior to December 12,
2012 and stated that Jinek 2012 had motivated their work. See Exs. 1557-1560.
77.
George Church has publicly stated that the work of Mali et al. was independent of
the Zhang lab of Broad. Ex. 1534, 67; Ex. 1535, 67.
78.
Research published within a year of Jinek 2012 used Type II CRISPR-Cas
systems in a variety of eukaryotic cells: human (Cho et al., Mali et al., Jinek et al.), zebrafish
(Hwang et al.; Shen et al.), mouse (Shen et al.), and yeast (DiCarlo et al.) cells. Ex. 1534,
63-68 and Appendix; Ex. 1535, 63-68 and Appendix; Exs. 1056-1060, 1372.
79.
Research published within a year of Jinek 2012 applying the Type-II CRISPR-
Cas system in eukaryotic cells used conventional techniques and reagents. Ex. 1534, 75; Ex.
1535, 75; Ex. 1055, at Fig. 1B; Ex. 1056, at Fig. 1A; Ex. 1059 at Supplementary Methods 2;
Ex. 1058, at Methods; Ex. 1060, at 720.
80.
Shuailiang Lin, a former Zhang laboratory member and a listed co-inventor on
Broads priority patent applications, admitted to Dr. Doudna on February 28, 2015, that the
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Zhang laboratory did not work [the use of the Type-II CRISPR-Cas system in eukaryotes] out
before seeing [the Jinek 2012] paper. Ex. 1475.
81.
Shuailiang Lin admitted that Feng Zhang and Le Cong quickly jumped to the
project to use the Type-II CRISPR-Cas system in eukaryotic cells after seeing UCs paper in
June of 2012.
82.
Fei-Ann Ran, a co-author of the Cong publication and one of Broads named
inventors, wrote:
When we started working on this, the tracrRNA hadnt been discovered yet. . . .
At that time, two other developments also emerged (1) you can fuse the spacer
and the repeat and the tracrRNA into a single chimeric RNA; and (2) you can use
a single chimeric RNA and Cas9 to program the cleavage of DNA targets in an in
vitro cell-free lysis reaction. We built upon these exciting discoveries.
Ex. 1561, at pp. 71-73.
83.
Fei-Ann Ran wrote:

obviously, bacteria dont have nuclei, whereas mammalian and other eukaryotic
cells do, and so we tagged NLS (nuclear localization signal) sequences to Cas9
and also codon-optimized it for better eukaryotic expression. By doing this, we
were successful in moving the Cas9 enzyme into mammalian nuclei.
Ex. 1561, at p. 73.

84.
Dr. Carroll wrote:

The authors [of Jinek 2012] make the bold prediction that this system can
potentially be used in place of ZFNs or TALENs for targeted genomic cleavage in
higher organisms. Lets think about how this might work.
Ex. 1152, at 1659; Ex. 1534, 31; Ex. 1535, 31.

85.
With regard to using a Type-II CRISPR-Cas system in eukaryotic cells, Dr.
Carroll wrote it is clearly well worth a try. Stay tuned. Ex. 1152, at 1660 (emphasis added);
Ex. 1534, 34; Ex. 1535, 34.
2-15

86.
Kent Golic stated that [i]t was immediately obvious that [the Type-II CRISPR-
Cas] system might be repurposed for genome engineering, similar to ZFNs and TALENs. See
Ex. 1473, at 306; Ex. 1534, 36; Ex. 1535, 36.
87.
Techniques one might use to practice the methods of UCs claims in eukaryotic
cells were well-known and routinely used in the art. Ex. 1534, 39; Ex. 1535, 39.
88.
Dr. Simons admitted that all of the techniques one might use to apply Type-II
CRISPR-Cas in eukaryotic cells, including those that are taught in Broads own patents and
application, were conventional at the time, and that a person of ordinary skill in that art would be
capable of performing the conventional methods. See Ex. 1555, at p. 97, ll. 19-22, p. 134, ll. 520 (techniques generally conventional), p. 146-153 (specifically referring to techniques
described in Broads provisional application Ex. 2101).
89.
Methods for introducing a nucleic acid into a eukaryotic cell had been well-
known for over 30 years. Ex. 1534, 44; Ex. 1535, 44.
90.
By 2012, it was well-known that a protein or nucleic acid, including those of
prokaryotic origin, could be expressed in a eukaryotic cell using an expression vector, including
viral vectors. See Ex. 1534, 44; Ex. 1535, 44.
91.
By 2012, it was well-understood that heterologous expressed proteins could be
targeted to the nucleus using one or more nuclear localization sequences (NLSs). See, e.g., Exs.
1029; 1235; 1236; 1319; Ex. 1534, 45; Ex. 1535, 45.
92.
One of ordinary skill in the art understood how to increase expression of
prokaryotic proteins in eukaryotic cells by codon optimization. See, e.g., Ex. 1030; Ex. 1534,
45; Ex. 1535, 45; see also Ex. 1315; Ex. 1576, at 796-98.
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93.
Directly injecting a pre-assembled prokaryotic protein/RNA complexes into
eukaryotic cells had been used. See, e.g., Ex. 1293, at 12; Ex. 1534, 46; Ex. 1535, 46.
94.
Prokaryotic systems had been used in eukaryotic cells for decades prior to 2012.
Ex. 1534, 52; Ex. 1535, 52.

95.
The prokaryotic Cre-Lox site-specific recombination system had been used
successfully in eukaryotic cells since the late 1980s. Ex. 1534, 54; Ex. 1535, 54; see Ex.
1335; Ex. 1336.
96.
The prokaryotic EcoRI restriction endonuclease had been used in eukaryotic cells
since the late 1980s. Ex. 1534, 56; Ex. 1535, 56; see Ex. 1302.
97.
The prokaryotic RecA protein had been used successfully in eukaryotic cells from
at least the mid-1990s. Ex. 1534, 57; Ex. 1535, 57; see Ex. 1329.
98.
The C31 recombinase from Streptomyces lividans was well-known to display
activity in mammalian cells. Ex. 1534, 60; Ex. 1535, 60; see Ex. 1327.
99.
DNA-targeting ZFN and TALEN systems utilize the prokaryotic FokI restriction
endonuclease domains to cleave eukaryotic target DNA. Ex. 1534, 52; Ex. 1535, 52.
100.
Sauer reported, in 1987, that a procaryotic recombinase can enter a eucaryotic
nucleus and, moreover, that the ability of the Cre recombinase to perform precise recombination
events on the chromosomes of S. cerevisiae is unimpaired by chromatin structure. Ex. 1534,
54; Ex. 1535, 54; Ex. 1335, abstract (emphasis added).
101.
Dr. Simons acknowledged that Sauer demonstrated the use of Cre-Lox
recombinase in eukaryotic cells. Ex. 1556, at 234, l. 21-235, l. 14.
2-17

102.
Reiss et al. demonstrated, in 1996, that the prokaryotic recombination protein
RecA itself is capable of interacting with genomic homologous DNA in somatic plant cells.
Id.; see also Ex. 1329, Abstract; Ex. 1556, at 240, ll.11-14.
103.
Dr. Simons admitted that he has not cited any evidence that any difference
between prokaryotic and eukaryotic cells has actually presented any specific difficulty in using
the Type-II CRISPR-Cas system and methods in eukaryotic cells. See Ex. 1555, p. 179, l. 8-p. p.
180, l. 9, p. 192, l. 10 p. 193, l. 16, p. 202, l. 2 22,. p. 203, ll. 16-21.
104.
Dr. Simons admitted on cross-examination that he did not have any evidence that
misfolding proteins was actually a factor or an impediment to the successful use of Cas9 systems
in eukaryotic cells. Ex. 1555, at 179, ll. 14-19.
105.
Eukaryotic DNA is not fixedly bound in chromatin, it was known to be a dynamic
system, constantly exposing different areas of DNA. Ex. 1534, 96; Ex. 1535, 96.
106.
DNA in eukaryotes is not limited to chromatin bound genomic DNA. See, e.g.,
Ex. 1556, p. 229, l. 16-p. 230, l. 15.

107.
Examples were known in the art of RNA molecules that could be expressed in
eukaryotic cells and functionally interact with proteins. See, e.g., Ex. 1229.
108.
Dr. Simons admitted on cross examination that each of ribozymes, riboswitches,
and self-splicing introns that he cited as examples, were actually shown to function in eukaryotic
cells in the references that he himself cited. See Ex. 1556, p. 216, ll. 19-20, p. 219, l. 21 222, l.
18, p. 225, ll. 5-13.
109.
Riboswitches, ribozymes, and self-slicing introns are not proteins like Cas9. Ex.
1534, 88-89; Ex. 1535, 88-89.
2-18

110.
A person of ordinary skill would not consider riboswitches, ribozymes, and self-
splicing introns to be analogous to Cas9 or predictive or its activity in eukaryotes. Ex. 1534,
88-89; Ex. 1535, 88-89.
111.
Wirtz et al. show that the T7 polymerase can access and copy eukaryotic DNA.
Ex. 2240, at 4626; Ex. 1534, 85-86; Ex. 1535, 85-86.

112.
UCs first provisional patent application demonstrates that UCs inventors
expected that Type-II CRISPR system could be readily adapted for use in a eukaryotic cell. Ex.
1534, 70; Ex. 1535, 70; see also Ex. 1003, at [00124]-[00129], [00165]-[00177],
[00186]-[00188], [00216], [00178]-[00179], and Figs. 1-4.
113.
In an interview published on June 28, 2012, Dr. Doudna explained UCs recent
discovery: Weve discovered the mechanism behind the RNA-guided cleavage of doublestranded DNA that is central to the bacterial acquired immunity system. Ex. 1546, at 1.
114.
In an interview published on June 28, 2012, Dr. Doudna stated: Our results could
provide genetic engineers with a new and promising alternative to artificial enzymes for gene
targeting and genome editing in bacteria and other cell types. Ex. 1546, at 1 (emphasis added).
115.
In an interview published on June 28, 2012, Dr. Doudna stated [a]lthough weve
not yet demonstrated genome editing, given the mechanism we describe it is now a very real
possibility. Ex. 1546, at 1
116.
Dr. Simons testified during his deposition, [o]ne never does an experiment
without the belief that it might work under certain circumstances. Ex. 1555, at p. 178, ll. 10-12.
2-19

CERTIFICATE OF FILING AND SERVICE
I hereby certify that on this 15th day of August, 2016, the foregoing UC et al.
OPPOSITION 2 is being filed, via the Interference Web Portal, by 5:00 PM Eastern Time.
Pursuant to agreement by the parties, service copies are being sent, via electronic mail by 8:00
PM Eastern Time today, to Junior Partys counsel as follows:
Steven R. Trybus, Esq.
Harry J. Roper, Esq.
Paul D. Margolis, Esq.
JENNER & BLOCK LLP
353 North Clark Street
(312) 222-9350
strybus@jenner.com
hroper@jenner.com
pmargolis@jenner.com
/Todd R. Walters/
Alexandria, Virginia 22314
Telephone (703) 836-6620
Facsimile (703) 836-2021
Counsel for UC and Vienna

UC Opp No. 2 CRISPR Patent

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Filed on behalf of Senior Party

THE REGENTS OF THE UNIVERSITY OF CALIFORNIA,

Todd R. Walters, Esq.

By: Li-Hsien Rin-Laures, M.D., Esq.

UNITED STATES PATENT AND TRADEMARK OFFICE

Interference No. 106,048

THE EVIDENCE .................................................................................................................2

STATEMENT OF MATERIAL FACTS.............................................................................2

Broads Sole Argument is That There Would Have Been No Reasonable

Broad Has the Burden of Proof ................................................................................3

Contemporaneous Evidence Demonstrates Motivation and Reasonable

Multiple Researchers in the Art Had an Expectation of Success in

Like All the Others, Broad Merely Made Obvious Modifications to

The Contemporaneous Quotations Cited by Broad Further

Examples of Successful Use of Prokaryotic DNA-Targeting

Interference No. 106,048

Broads Arguments are Unsupported and Contradicted by

Broads Arguments of Non-Specific Differences Between

Broads Non-Analogous Examples Would Not Dispel the

Comments of UCs Inventors Do Not Contradict the Reasonable

APPENDIX 1 - LIST OF EXHIBITS

Interference No. 106,048

Alarm.com v. Icontrol Networks, Inc.,

Interference No. 106,048

and claimed by Senior Party (UC) in eukaryotic cells.

Interference No. 106,048

STATEMENT OF MATERIAL FACTS

Opposition are also set forth in Appendix 2.

CRISPR-Cas system in a eukaryotic cell renders Broads claims separately patentable.

Broads Sole Argument is That There Would Have Been No Reasonable

Interference No. 106,048

successfully function in a eukaryotic cell.

5-10; Ex. 1535, 5-10.

Broad Has the Burden of Proof

Interference No. 106,048

Broad has completely ignored, or failed to rebut, overwhelming evidence of obviousness,

reasonable expectation of success of implementing a CRISPR-Cas system in eukaryotic cells. At

eukaryotic subject matter of Broads involved claims obvious.

provisional application. Ex. 1534, 18-32; Ex. 1535, 18-32.

Interference No. 106,048

contemporaneous reactions of those of ordinary skill in the art.

2012 suggested using the Type-II CRISPR-Cas system in eukaryotes, stating:

Recently, Jinek et al. (2) elegantly demonstrated that a single-chain chimeric

CRISPR-Cas system in eukaryotic cells, one of which is summarized as follows:

See, e.g., Ex. 1010, cover sheet.

Interference No. 106,048

the Type-II CRISPR-Cas system recited in UCs claims.

Interference No. 106,048

105; 119-120; 244-251; 277-282), or supplying a Cas9 site-directed modifying polypeptide to

available to do so (including expression vectors, nuclear localization signals, and codon

0042, 0054, 0058, 0060.

issued as Broads involved 359 Patent. Ex. 1601, at 3-16.

Interference No. 106,048

type of cell, including eukaryotic cells.

Jinek 2012 concludes with the following proposal:

Zinc-finger nucleases and transcription-activatorlike effector nucleases have

(ZFNs) and transcription-activatorlike effector nucleases (TALENs) were, at the time of

Interference No. 106,048

Jinek et al. realized that a highly specific, customizable RNA-directed DNA

commentary accompanying its publication demonstrate a high expectation of success that

evidence, Broad further failed to meet its burden of proof.

Contemporaneous Evidence Demonstrates Motivation and Reasonable

Interference No. 106,048

applying a Type-II CRISPR-Cas system in eukaryotic cells required overcoming uncertainty