IN THE UNITED STATES PATENT AND TRADEMARK OFFICE,
In re application of: Confirmation No. 8182
THE REGENTS OF THE UNIVERSITY OF Examiner: NOT ASSIGNED
CALIFORNIA et al
Technology Center/Art Unit: 1636
Application No.: 13/842,859
Filed: March 15,2013 THIRD PARTY SUBMISSION
UNDER 37 CFR. $ 1.290
For: METHODS AND COMPOSITIONS FOR RNA-
DIRECTED TARGET DNA MODIFICATION. .
Customer No.: 115985
Commissioner:
The Examiner is requested to consider the following remarks regarding:
WO 2013/141680 (cite no. 1) as they relate to pending claims 1, 2, 5-8, 10-19, 22-
25, 29-35, 38-45, 48-52, 56, 58-60, 64, 65, 73-77, 84, 86, 89, 102-106, 109-111, 113-121, 148,
and 149 in US 13/842,859 (the '859 application);
Sapranauskas er al. (cite no, 2) as they relate to claims 1, 2, 6-8, 11, 1214-17, 38,
40, 44, 45, 49-52, 56, 64, 65, 74-77, 109-118, and 121 in the '859 application ;
Gameau er al. (cite no. 3) as they relate to claims 1, 2, 6, 11, 44, 45, 49-
56, 64,
65, and 74-77 in the '859 application;
Notice of Allowance and Fee(s) for US 14/054,414 (cite no. 4) as they related to
claims 38, 40, 41, 43, 64, 69, 70, 92-95, 98-102, 104, 122, 130, and 131 in the '859 application;
Mali ¢7 al. (cite no, 5) and Cong er al. (cite no, 6) as they relate to claims 38, 40,
64, 69, 70, 92-95, 98-102, and 104 in the ‘859 application;
WO 2014/065596 (cite no. 7) as they relate to claims 38, 40, 41, 43, 64, 69, 70,
92-95, 98-102, and 104 104 in the *859 application; and,
WO 2014/089290 (cite no. 8), US 8,697,359 (cite no 9), and WO 2014/099750
no, 10) as they relate to claims 38, 40, 41, 43, 64, 69, 70, 92-95, 98-102, 104, 122, 130, and
13 in the '859 application.
The remarks cite to the paragraph numbering of the '859 publication, US 2014-
0068797 Al (the "797 publication).Application No: [3842859
‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014
Page?
WoO 2013141680 (Cite No.1
CLAIM t ‘WO 2013/141680
Claim 1
As scusedon p. 23, ns 1630, WO 2013141680 discloses
NAsrrRNA compl iin cope of DNAageing RNAS seed
ADNAsupeing RNA comping in claim and defined at [0136] & [0137] ofthe "97 publition.
‘The DNA rgting RNAS st fat in WO 2013/4168 ince many of he
same element discussed inthe peiication ofthis pplication,
(i) aft segment comprising «ule segue tt complementary oa
sequence na target DNA and
DNA arging RNAS having segment conplemenay oa sequence na
‘target DNA of elim | iene dslosed a. Fgue 20C and tp, 2,
lines 79a pe 24, ines 1-28 of WO 2013140
Figure 20 (iscusd on, ines 14.28 discloses a crRNALacRNA
copes having a paver egion complementary oa sue ina target DNA
and. ines 7-9 discloses [ interne gis phage end plaid DNA
wove by therapies CRISPR3 runes te presence, within the trget
DNA, ofa rotospuoersouenoe complementary tothe pared
rRNA?
ia seond segment that inkras waste dicted mdiling polypoid
DNAsagetng RNAS having a cond segment tatters wth te-
diet ming polyepie of cain |isexpesydsloedat eg Figure
20 of WO) 201314680, which ses eRNA.trceRNA compled wih
Oss,
“Site dread motifing poppies wed inca Lief in
paragraph [O18] ofthe 797 pubation inclu Ca.
Cain
The DNA tating RNA of cli |, wherein he fist egmen comprises 8
ruckoides tt have 1 complemen a equ in th tget DNA,
The fist segment comprises ® mci tat ave 10% complement to
a sagen inthe tet DNA ofl is dled ate, ci 24 of WO
2013/4168 wc ceted oe protspacer sequence ina target DNA tat
is 10% complementary ta 20-mcetide spacer equnce ina GRNA.
Chaim $
The DNAtapeting NA of cin |, when tested moijing
olypepide compris en nina acid equnc having teas about 759%
WO 2013141680 cisloses DNA-trgeting RNAs having at east about 75%
anno cds guen: ident oth conesgnting potions of SEQID NO:Application No: [3842859
‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014
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WO 2013/141680 (Cite No. 1
CLAIM
‘WO 2013/141680.
‘amin acid sequence ety to amino acs 7-166 31-103
C9 ain acd sequence deen 1G 3 0 tothe coresponding
tons in ny ofthe amino acid sequences set orth as SEQ ID NOs I-56
and BEM,
1317. SEQUDNO: [in WO201374 680 as rete tn 107 iio
‘SEQID NO: 1317 in claim §,
Claim 6
ADNA polynckoide comprising a mule eee tht ene the
DNA-eting RNA of in 1
The DNA plynucetide ending 2 DNA-trging RNA of claim 6s
islet a eg, page, ines 2-24 and Fi. LB of WO 201314168, which
isles pCa) has sequencing encoding DNAagetng INA
(vacRNA ad SP)
Chaim 7
recombinant expression etrcuprsingte DNA polymeleie of claim
4
The eonbinant epeson vector comping aDNA poljrckide
cncing a DNA-rgting RNA of lai Tis lose seg, pines 2-
Mand Fig. [of WO 201341680 which dla cheat eresetation
fifrlogous iin two plasmids use farthe aversion of the Cas8-
crRNA complex pCas SP isan expression vector with souenoes noting
4 DNAHtreting RNA (ra RNA ad SPI),
Chaim 8
The reombinant expression etr of cm 7, ween the aloe
sequence encoding the DNA tretng RNA i operably linked toa prom,
The vcr in wich te aukoide eqeneeeoing te DNA geting
BNA isoperty ike a promote of cin 8 isso at 3 ies
30.32 and claim 17 of WO 201/14168, wich dose ht the components
ofthe Cas complete (ineuing ar RNA and erRNA) en be in plasmids
containing bt promote),
Claim 1)
The reoombinan! expresso eir of chim 7, when th uli
sequent econ th DNA-tgtng RNA of cai fuer comprises 2
ull loring si,
The vectors iter conpsng mull cloning sites of claim 101 isosed a,
25. fines 17.22 of WO 201314168, wich dsr the cloning of
CaSPI psi,
Chim 17
Ain vito genetically modified host cel omprisng the DNA plynuclie
The geticlymolied hs cel comping DNA polytuleoties noting
DNA ering RNA of chi 1s ck at pI ies 3.27 of WOApplication No: [3842859
‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014
Paged
WO 2013/141680 (Cite No. 1
‘WO 2013/141680.
of claim 6,
141680, which discloses how the pCasi-)SPI plasmid was introduced
into E.coli cells,
‘Chim 12
resin expression et comprising:
WO 213761680 dso reconbiartexeson vero comprising
racleotde sequences encodings DNA tein RNA anda sie dited
ding polypeptide. Se, eg, .3ines 3032, wich disclose thatthe
components fhe Cas compeesincting Cs), racrRNA, and rRNA)
canbe iin sng pls. Sea 9.29 (claim 17,
(Tena segue ending DNA ring RNA, when DNA-
‘aretng RNA comprises: 2) fit segment comprising a mucleotide sequence
‘atiscomplemenry toa sence ina tare DNA; nd) 2 second
segment hat inka wha ste- dred ming polyp and
‘WO 2013141680 does uke sequences encoving DNA tating
RNAs. Se dssson facia,
(ianuckaide equne enoning the ste-ieted modifing olypeptibe
comprising: aan RNA-binding prion hat inert with the DNA targeting
NA, and (nativity potion hat existe dee enya ati,
herein he site of enymati atvityis deterine bythe DNA-tgetng
RNA
TWO 2013418 dls ecnbinan exesion eos comprising
rote sequen ening Cs in atone nuckeie sotence
cnowinga DNAirgtig RNA (eg, cRNA and tracrRNA).
“Sie dese ming poypepli” as wed in cli 12s defied in US
214 M6STST io incude Cad.
Claim 13
A revonbinat expression vector omprnng:
‘WO 2013/141680 discloses recombinant expression vectors, See discussion
for claim 12, above,
(Tanai seguro endings DNA ring RNA, when heDNA-
‘ewig RNA copies 2 fis seen comping mcletie sequence
ttatiscomplemeniry toa sence inate DNA; and) 2a
segment hat interes waste dred ming polyp and
WO 2013/41680 dco rexombinantexpeson vcs with ule
sequences ening DNA geting RNAS, Se discussion for cai 2 aoe
(ive ruckoteseqpne eng te se dieed modifi ope,
heen sede ming polpeide compris: (2) an RNA-
binding portion that interacts withthe DNA-tretng RNA; nd (ban activity
gorio tt mois rnsriionwitin heft DNA, when the se of
modulated transcription within the target DNA is determined by the DNA-
WO 21374168 dsloses olenide sequences encoding se decd
uahng polypepies that ave an RNA-binding portion at ners with
DNAdaptng DNA and an att portion tat modus transcription
within te age DNA, Seep, 1 ines 23, which ists Cas) mutes
in which th endaeanyrbonuckase acy ised he mutans canApplication No: [3842859
‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014
PageS
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‘WO 2013/141680.
ving RNA
lave ly one stand sie of bh sands)
Vacant Cs st-ieted polyps eli redued
endodeoyrbomukase atte infued an exangl of sided
nding polpepties wih navy portion that mules arson of
target DNA in paragraph [0409] of US 201410068797,
Chaim 1
A variant site ieted mailing polpeplide comprising
(Daa RNA inking portion tira whe DNA ageing RNA,
‘be DNA trptng RNA comprises aac sequence hat is
complementary ia eqn ina target DNA and
(i) anactivty potion hat exhibits roducedserected enzymatic activity,
vein te si of ext activity i dein hy th DNA rting
RNA.
Wo 2013141680 ciscloes variant se reced mating polypeptides. See
discussion for claim 13, above,
Chaim 15
‘The variant steed modifying polypeptide of cam 14, comprising an
S404 matation oe. pyogenes sequence SQ NOS orth
conespnting maton in any of te anno acd sequen se fthasEQID
‘NOs:1-256 and 795-1346.
WO 23161680 does variant sie rected modi
comping a coreg tion n SEQ ID NO
Lines 23-2 (closing Cas mutants with tied HN aie temo,
exemplified by NI91A and 868A). These mutations ge he same ative
site (HNH) as HRAUA,
‘The unmutatd Cas) soquence in WO20L3 141680 (SEQID NO: 1) is 10%
identical in quence SEQ ID NO: 31 inci 13.
Claim 16
The aint se dred mong pole of cm 4 comprising
DIOA mutton oft. pyogenes sec SEQ ID NO athe
comesponding mutation in any ofthe amino aid sequenses set fot s SEQ ID
Nos 26 and 95.1346,
WO 213141680 dloes vat ste deed moilying poppies
comprising acoresponing mtn in SEQ ID NO: 1317, Se, eg, page 3,
Lines 23-2 (closing Cas mata with muted Ran ative site mi
exemplified by D31A), Th D31A muti tages the same ate site
(RuvC)asDI0A.
‘The unm Cas) soquence in WO20L3 141680 (SEQ ID NO: 1 i 10%Application No: [3842859
‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014
Pages
WO 2013/141680 (Cite No. 1
‘WO 2013/141680.
iden a sguence to SEQID NO: 1317 ncn I.
Claim 17
17 The var sidered moda polypeptide of clin 4, comprising
bath
WO 2013141680 ciscloes variant screed moaving polypeptides. See
discussion for claims 15-16, above,
(De DIGR maton of eyo sequence SEQID NOR ie
conespnting maton in any of te anno acd sequen se fthas EQ ID
‘NOs:1-256 and 795-1346; and
WO 2013141680 dloes vat ste ted moda polypeptides
faving tations coreponing to DIDA. See discon for cai 6 above
(Gyan RDA mutton oe S,pyogns sequence SEQID NOS ort
comespening mutation in any ofthe anno acd sequences seth as SEQID
NOs 286 and 951346,
WO 2013147680 discloses varia ste directed modlyng polypepies
faving tations corespening to HBAIA, Se dscson fe claim 15,
abe
Chaim 18
‘A cher se dieted nding polypeptide comprising
(Gan RNA Finn potion tat incr wit a DNA arpeing RNA, when
the DNAtargtng RNA comprises anceidesequne hati
complementary ia equne ina tage! DNA; and
(ian activity potion tates dietdeazymati activity, wherein
these ofenzmati atts deine bythe DNA targeting RNA
WO 2314168 does chinersite-deed moi ying polypeptide,
See, eg, ll nes 12 (een steed Cs and Hind
Cas9), See also discussion for claims | and 12, above.
“Chiner polyp” df in paragraph 0115 of US 201406897 a
include polypeptides made by the combination isin of to obese
scare seen of anno acids.
Claim 19
The chime sired ning polyp of cai 18 compesing en
amino aid sequen having tes about 75% ain aid segue identity to
amino wis 71660" 731-1018 ofthe Cs9 Cs anno ac sequene
depicted in 1G. 3 orto the corespoting ports in ayo tai aid
sequen sf 8 SEQ ID NOs 286 and 95.134,
\W0 20131468 dls chines se deed nding poy
avingat est 75% eqns ident o oto mor of he SEQID NOs in
claim 19. See discussion for claim 5, above,
‘Chim 22
‘The chimeric ste ined modiyng polyepide of lai 18, wherein he
eosyatic activity modes the tut DNA,
‘WO 2013/141680 discloses site-directed modifying polypeptides with
aya ay ht mites tet DNA, Se, 9.23, ies 31.32, and
(p.24, lines 7-24 (disclosing how Cas9-crRNA complexes cleave both strandsApplication No: [3842859
‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014
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‘WO 2013/141680
af rg DNA ate ses)
‘Claim 23
The chime sided ning polypeie of din 2, wherein te
earynatic att is ruckase acy, methane ety, demety ase,
activity, DNA npuiraiviy, DNA damage cy, deamination att,
cistae avy, alain avy, depuration avi, oiatin ati,
pyvinidn dime forming atv, integrase avy, tansposis acti
evonbie avy, polymers ati, ie atviy, bela atviy,
phos ati or gieospae acy.
‘WO 2013/141680 discloses chimeric site-directed modifying polypeptides with
nuclease activity, See discussion for claim 22, above
Chim 2
The chimeric stedinedmodiyng polypepide of lai 23, wherein he
enzymatic activity is nuclease atvity.
WO 2013/4160 cisloses chimeri sie-recied malying polypeptides with
nuclease ati, See discussion far claim 2, ave
Chaim 25
The chime seine lng polyeide of din 24, wherein te
rules at inte double ste bein th target DNA.
Wo 2013/4680 ciscloes chimeric sie-reced mealying polypeptides with
double strand nuclease activity. See discussion for claim 22, above
Claim 29
‘An RNA polymuclide comprising 2 ruled sequence encoding the
chimeric eed modiljng polypeptide of im 1,
‘WO 2013141680 discloses RNA comprising a muclvide sequence encodings
cher site-directed moilfngpolypepi,Se, eg, pI nes 7-12
(disclosing step aggod Cas) and éxBistaggd Cas). RNA is necessary an
ferme sp in pring he Cs) pron om he pASKIBA3 Cs)
les orth pBAD Cas pls
Claim 3
‘ANA pljuckotide comprising alee eee encoding the
chime site-deved mying ply of cin 1,
7 WO 20031660 dls DNA peracid ecdng chines
ied lying polyps Se sus or cin 9, hove.
Claim 31
A resoinant expression elor comprising te plymuletibeof im 3,
‘WO 2013/141680 discloses expression vectors comprising DNA
polynucleotides encoding chimeric site-lirecied mifying polypeptides. SeeApplication No: [3842859
‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014
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‘WO 2013/141680.
«iscassion fr claim 29, above,
‘Claim 32
The oben expression vert clin I, wherein he poynaties
openly Hn apron
‘WO 2013)/141680 discloses expression vectors comprising DNA
rely ening chine sired odin olpeptites,
whith pluses nko promo See dscusin ein
29, above.
Claim 4
Ania vio ently mdf ist cell comprising the plyncetie of
claim 30
7 WO 20134160 dls eal mold cel ompsnga DNA
oly ecg a chimeric seseted moding poppe. Se,
gpl ies 3.2 (dkxng geeticaly mie Ecol el comping
less exesng Cas,
Claim 35
chimeric stedeed malin olpepie comprising
(ax RNA ining portion fat inert with a DNA argeing RNA, wherein
the DNA arptng RNA comprises amide sequence hat is
complementary ta equnce ina ret DNA, end
(i anactviy portion tat modulates taneiion within te txt DNA,
vente seo mde tans win te txt DNA is
celermied by the DNA taretng RNA.
WO 23141680 dsloes cinerisie-dresad nding polypeptides.
Seecisusion or clin 13 and 18 ahve
Claim 38
A geaclly mdf cel omprsngeeobiant sie etd modifi
polypeptide comprising an RNA hndng portion tt inert witha DNA-
tei RNA; nd nai prtion tat exbissedietd engi
activity, when te sie of erzymati activity determine bythe DNA-
‘eng RNA
WO 2013141680 discloses genetically modified hs cel comprising DNA
polynucleotide encoding a recombinant site-dievted modifying polypeptide.
Se, eg. pl ines 3-27 (sling genetically moti’ Ecol els
comping pls exesng Cis).
‘See also discussion for claim 12, above.
Chain 39
The gncticaly moe cell of claim 38, wherein the stedieted moifving
‘Wo 2013141680 cisloses maf cells with site-directed modifyingApplication No: [3842859
‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014
Paged
WO 2013/141680 (Cite No. 1
CLAIM
‘WO 2013/141680.
polypeptide compries en amin aid sequence ving es cout 5%
amino aid sequence ety to amino ci 7-66 cr 3-108 of ti
asl amin acid equnce dict n 1G 3, or the omespoding
porios in any ofthe amin acid sguences st arth 5 SBQUD NOs 256
and S136,
elpeties avn atest 75% amino ai sqoence ety tone ore
cof the SEQ ID NOs in claim 39, See discussion for claim 5, above,
Claim 40
‘The genetically modified cel of claim 38, wherein the cel is selected from the
oup consising of: an archaea cel hater cell, a eukaryote cel a
clan anal el in invert cel verter cela sh
celhafrgcel aid el a mammalian el piel, cow el gat cl,
a sheep cel, a rodent cell, a rat cella mouse cel, a non-human primate cel,
anda hun cel
ell organism, a somatic cl, or cel a tem cell plat
‘WO 2013/141680 discloses E.coli cells. See discussion for claim 38, above,
Claim 41
A ragenicnor-bunan organi wins geome comprises ansgene
comprising a uct sequen encodings ein! ste diet
odin polypeptide comping
{an RNA ining portion eras wit a DNA engin RNA; and
(inactivity potion tt exis sede emma activi, erin
these ofenzmati atts dlerined bythe DNAcargting RNA
WO 2013/4160 cisloses transgenic E ol cel, Se discussion for ims
12 and 38, above,
Chim 22
The tangeni organism of clin, when the site-ieed modifying
polypepide compris en amino acid seguence having st es about 3%
amino aid sequence ety tain ci 7-166 cr 3-108 o th
Casal ain acid equnce depicted n FG, 3, or the comping
orion inany ofthat acid sequences set fora SEQID NOs 256
an B14,
WO 2013141680 discloses sit rected odin polypeptides having at
{east 5% identity to one or more ofthe SEQ ID NOs in claim 42, See
dicusion for claim 5, above,
Chaim 43
Te ranger rio of ci 1, when the anion seed on
‘WO 2013/141680 discloses transgenic E.coli, See discussion for claim 38,Application No: [3842859
‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014
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WO 2013/141680 (Cite No. 1
CLAIM
‘WO 2013/141680.
ihe wou omsitng of an az, haven, ekayoie sige
nga, an alga plant, an anna, an invert, fa wom,
rian vert afk fg bil anal, an ung, rk &
fe mouse and nonhuman priate,
above,
Chaim
A camposton opr
WO 23141680 does composton comprising DNA-agting RNAS
and site-dneced modifying polypeptides. Se, 2.23 ines 1622
(demorstating comple comprising Cas, RNA, and raiRNA), Se ao
28cm},
(i) a DNA targeting RNA, or DNA polymucleoide enonding the same, te
DNA-treting RNA comprising (ast segment comprising aclie
sequence tt compemenary ia seen in art DNA; and) 2
send semen hat ina Wise deve non pope nd
‘WO 2013/141680 discloses DNA targeting RNAs, See discussion for claim |,
above,
(i ese ded ying pol pede, or apolaleid enodng te
sane te ede ming polypepide comprising (2) an RNA-binding
eotion tht interacts with the DNA tareing RNA; an (b) an atv ption
hates sere enzymatic activi, when te sof enya
axis determine bye DNA tagetng RNA.
\WO 20131468 dls steed mong polpepies, See
ciao fr cans | nd 12, above,
‘Chim 45
The composition ofan 4, wherein te ist segment of th DNA aging
NA comprises 8 mcletdes that ave atest 10s complementary oa
sequence inthe tpt DNA,
WO 2374168 doses composton comprising DNA-agetng RNAS
and sie-dneed odin polyenes, wherein the fir set fhe
DNAagtng RNA bs 100% complementary toa sequace in he gst
DNA, Ser, ep 30 (lim 24)
Caio $8
The composition of cin 4, wherein the ie etd nding polpepide
compris an ain acid sues having! ast shot 7% nina acid
sequence iden toaming ais 7166 731-11 of he Cash Cs ang
‘cid seer depicted in 1G, 3, orth oman prions nny the
ain aid eguanes et fos SEQD NOs 256 and 795-1346,
WO 20151468 dls steed modying pobpepieshvig at
let 15% sequence ety toner o te SEQ ID NOs in chim $8
Seeisasion fr cin ato.Application No: [3842859
‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014
Pagel
WO 2013/141680 (Cite No. 1
CLAIM
‘WO 2013/141680.
Claim 49
The composition of cin 44 wherein te enzymatic actly mos he
tay DNA
7 WO 200314168 discloses enzymatic activity tat mies ret DNA
through double strand breaks, See discussion for claim 22, above,
Chaim 50)
Te conposton ofan 4, wii te ent acy aks
ax, mettre activity, deme aiiy, DNA reir,
DNA dane ay, dant tivity dsm ctv, alkylation
acy, depuration avi, oxidation activi primi dimer oning
any, ilerase acy, assay, reombinse avy,
polyesters ay, poly atiyor
lynsey.
‘WO 2013/141680 discloses enzymatic activity that modifies target DNA.
‘rough double stand beats, Se disusion or claim 22, above.
‘Chim 31
The conpostion of cin 5, wherein te enzyatc ait is mulease
ati.
Wo 2013/4680 discloses enzymatic att tht moe trgt DNA.
‘through double strand breaks. See discussion for claim 22, above,
Chim 52
The composition of ci I, wherein fe muck atv intodues dub
strand brent get DNA,
WoO 2013141680 cisloses enzymatic activity tht mois target DNA
‘through double strand breaks, See discussion for claim 22, abywe,
Claim $6
The composition of im 4, wherein the DNA areng RNA fsadbl-
molecule DNAtargeting RNA andthe composition comprises bh 2 tareter-
RNA and ancl RNA the dpe ming sean f which re
complementary and yt frm he second seen ote DNA-
‘targeting RNA,
‘WO 2013/141680 discloses double-mokecule DNA-targeting RNAS, See
casio fr clam above.
Chaim $8
A conposion comping
{DDNA ageing NK of ci ora DNA plc encoding he
same and
WO 23761680 dos compostons comprising « DNA-argeing RNA
ana sbiliing bull. Se p23, ines 4-28, shoving amie of Cus,
crRNA, and taerRNA in but. Se as dscsion orci 44 core,Application No: [3842859
‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014
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WO 2013/141680 (Cite No. 1
CLAIM
‘WO 2013/141680.
(i bute for sizing mci ais,
Claim 59
‘AN composton comprising
(te sited modiyng polypepide of cm 4, ra pyaucetide
enon te some ad
(ia herr sbiliring mackie acids andor pris
WO 2013/6168 dios compostons comprising a ste diet ming
rlypeptiea a sailing bull, Seep, nes 4-8, showing a mite
of Ca cRNA, and taerRNA inate, Seals dsason forclaim 44,
abo
Chaim 6
‘A conposon comping
(DNA ring RNA ora DNA purl ending he sane, ie
DNAtupeting RNA comprising: (ist semen comprising a ruckotde
seqoence tit complementary i sequence ins gt DNA; nd) 2
second semen hat interac wiha sede ng pope and
(i este deed lying pol pede, ora polyale enoding te
sane, the sie-dreed odin plypepide comping: (2) an RNA Hndng
orion ht iets wit the DNA tarting RNA nd) an vty pron
‘bat moles tars wii te ere DNA, wen ke ste of
module tansspion within te tget DNA is eine byte DNA-
tugeig RNA.
WO 2013141680 dloss compostons comprising « DNA-areing RNA,
anda tit mdihing polypeptide that modules tension within
thet DNA. Se discon for cans 13 nd, hove
Chim 6f
Amst ose pce modisation oars DNA te metod
comprising contacting the get DNA wit
{DNA -argtng RNA, ora DNA pljncelbeencoding he sane,
‘hein th DNA rei RNA comp (2 fis semen comping
ruck sequence tts complemen inthe tet DNA;
and) ond set hikers wih ase dete modifying
polypepiea
(ipa sie-Tecedmoljingplpepie, ora pluton te
same, when thst modi poypepiecompis (2) an RNA-
WO 20131468 dloese matt see mtn of tpt
DNA comprising ccoing th target DNA wih a DNAreig RNA and
siete modingplypeptite Se, eg, 9.28 (ci I), See asop. 24
tins 7-14 (emonstating sie specication oa DNA doplex by nit
vi assembled CasrRNA comple), Se ao discs foci 4,
aboApplication No: [3842859
‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014
Page 13
WO 2013/141680 (Cite No. 1
CLAIM
‘WO 2013/141680
Binding prio tt inact wit fe DNA-areng RNA; ed () an city
portion tat exhibit diel enzymatic acti,
Chaim 65
The method of claim 64, wherein the target DNA is extacbromosoma
‘WO 2013/141680 discloses extrachromosomal target DNA. See discussion for
claim 64, hove, The DNA duplex iextracromnsomal
Chim 73
Themed of cam 4, wri fe DNA-odiing pol pepe compris
an amino ai sequence avingal st chat 73% amino ac sequence
‘identity 0 amino acids 7-166 ot 731-1003 of the Cas9\Csnl aminoacid
seqence depicted in FIG, 3, ofthe eoregoning portions in any ofthe
amino acid sequences set forth as SEQ ID NOs: 1-256 and 795-1346,
Wo 2015141680 doses DNA sniyng polypepdes having at ast 758%
sequence identity oon or nae athe SEQ ID NOsin lim 73, See
RNA) and anacivatr RNA (ie,
texrRNA), Sach mmpostion ae inherent preset for example, inthe
colcls crying pCRISPR closed on, 927, co, st rege
Claim 04
Amend of sie-specic mditcation of tet DNA, te etd
comprising contacting te target DNA wit
Spank eal dloes methods or sitespctic modification oa ret
DNA eg. p8P pls DNA) by coneng the tre DNA witha DNA-
targeting RNA anda ste-dreved modilying polypepide. See p.9280 (“We
‘show here that CRISPR3\Cas module cloned into E.coli is functionally active
and provides st liners aun plasmid and pn”)
(a DWAtrng RNA, ora DNA poljaucetde eooding he ane,
‘when te DNA argting RNA compris (2) first segment comprising a
rule sequence tats complementary a sequence in th tg! DNA;
and (ba send erent ht interacts witha ste iret modilying
polypepieend
Sapna dcloes DNA targeting RNAS. See discussion or chim
J,ahoe,
(istered mol plypepide, or polyrucide neding te
same, when thst deve modifing polypeptide compris: (2) an RNA-
binding portion ta inert wih he DNAating RNA; and (6) an acviy
orion ht existed enya ati.
Sapraaskaset al dls ste eed modihng oles. See
discussion for claim 1, above,
Chim 65
‘The method of claim 64, wherein the target DNA is extrachromosomal,
‘Spraasla eal dls target DNA thas extacnensomal, For
example, pSPl/pSP2 plasmid DNA an phage mia DNA (ue, 9.
9278) bsh examples of extairomnsomal DNA,
Chim 14
The method of cain 64, enzymatic activity modes ae
Claim 75
Sapraaskas tal dls enzymatic tite edb stand aces
eetvits)ht my tuget DNA, See cso for chins 4952, abe,Application No: [3842859
‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014
Page 26
‘SAPRANAUSKAS et al, (Cite No, 2)
CLAM
‘SAPRANAUSKAS etal.
The metnd of clan 4 when he enzymatic acy sue acti,
ety acy, dete atvity, DNA rei atity, DNA
damage aviv, deamination att, dims att, alkylation ati,
¢epurinion avy, oxidation ati, pyridine dime forming atv
negra atv, rerspsise avy, eombitase acviy, polymerase
activity, igase atv, bela avy, photolyase ato cose
actly
Chaim 76
Themed of cain 75, wherein ie DNA- nding enc acy
uclease activity.
Claim 17
‘The method of claim 76, wherein the nuclease activity introduces double
stand breakin te tert DNA
‘Claim 109
Akitconpeing
(the DNA ageing RNA of dim I, ora DNA ply ceoding he
saa
(Gj acaent fr econsiuon end dition
Claim 110)
The kit of clam 19, ier comprising reagent selected rom by oop
consisting of br for nroduing int el the DNS targeting RNA, 2
sash buf, contol eget, acon exesion vec or RNA,
polyno, areget or tanstng the DNA-arting RN frm DNA,
and combinations thet
Sapraaskas eal provides plaids encoding DNA ting RNAS
angen fr dion rules fr ntdton intel, See "Paid
‘rasformatios ection pacing pp. 927677, disclosing aCe solution for
inti th CRISPRS plasmid it oh
Cains 111-18, 21
See discussion for claims 109-110, above,Application No: [3842859
‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014
Page 27
Chim GARNEAU ea
(ameau eal disses DNAareing RNA, See alo p67, co.
ADNAMretng RNA comprising (Gsosing how “the CRISP Cas stem tags itherinvang DNA oc
RNA” and how this occurs through CRISPR RNAs (crRNASs}).
(ais seenent comprising aru equne tt i complanentry toa
sequence ina target DNA; and
Garrat. ses DNA get RNAs tae completly toa
sequence ina target mck ai. Se 67 co. Caspr the
RNAS to tage! foreign RNA by complementarity).
(i). ond segment thinkers wi se etd mdling polypeptide.
Garneau al sos DNA ring RNAs Tat neat wih sede
odin plypeices. Se p67, | (‘Cas protis use th er RNA o
tet eign RNA by complement’).
“Sie decd modiyng polyp” as wd incl |is dfnd in US
201410068797 inde Cas) (se prareph 0188). Cas and Cs ae
sh ames for Cash.
Chaim?
‘The DNA-targeting RNA of claim 1, wherein the frst segment comprises
ruck tt have complementary sequen inthe tet DNA,
amen adios DNA ugeting RNA bang segment bang
10% omplemetry to pNT1 target DNA, See. 67 cl 2: Sequence
anajsisof CRISPI nth elorenentoned 30 clos denied 14
fren spacers (435 al of which were enologus to pNTL
sequen (able | and Fg.”
Chim 6
ADNA polyno comprsinga mle een tht ene he
DNA-tagting RNA of ei |
Garneau ta. dsdoses DNA polauclenies ending DNA urging
RNAS (eg, cRNAS. Se pg 67) Sequence of CRISPI inthe
afemestioned 30 clones iden eet spaces (85-85) al of
hich were bomnlogns to NT guns (Table and Fig 1)”
Chim 11
Ani vi ently modified ist el comprising th DNA plynucetie of
cline,
Gently moi el comprising the DNA ening the cRNA were
created when the pNTLvetorwaslerparced int te thermos
cel Se poe 67. The intoduction ofthe pNTI vex geeialy
of th bast cel
Claim 4Application No: [3842859
‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014
Page 28
A compton comping
Garneau al diss compostons comprising DNA-ugeting RNAS eg,
crRNAs ands mdilyng polyenes eg, Cas pr. Se
1.67cok | sing bow “the CRISPRCa ste gt iter invading
DNA or RNA" and explanng bow Cas roti ws thc RNAS i tpt
feign uk acidsby conplnent).
See also p, 70 (“Overall we have established that the S. thermophilus
CRISPRI Caste caves bth aceriotage and planid DNA ins
Ts endonuclease acy, wc sens toe ous, prosper
spec an orton depend
CasSisambername for Ci), Se, eg, Cite No.2: Sepranaskas eal p
26,1 (a (amen nae aad exe)
(a DNAtareng RNA, ora DNA polyruclide enondig the sane, te
DNAstgting RNA comprising (ais seanent comprising a racket
sequence tas complementary oa sequence ina target DNA; and) sezond
segment thinkers wt iirc mang poled nd
areata. dsdoses DNA-agtng RNAs, See dun foci
above,
(i) the site-iexted modifying polypeptide, o a polynucleotide encoding the
sang, the sie-dreced modi plypepide comprising (2 an RNA tndng
porin tha nerats wih the DNA arting RN; and (9) an act potion
‘ht exits site diet enzymatic att, wherein sito enzymatic
activity detemind byte DNA geting RNA
Ganau etl dhs seid lying poppies, Sedan
fori, sbve.
Caio 45.
The compsition of ci 4, wherein he ist egment of the DNA rgting
NA comprises 8 nocletdes that ave atest 10s complementary oa
sequen inthe tug! DNA,
area a. discloses segmenting 10% complet tre
DNA. Session for eli above
Chim 49
The compton of ain 4, when te enya avy modes the ret
DNA,
Claim $0)
Garneau ta diss wonpostion He toe nein that kave
endomuclesatit that mdf taget DNA though double tand
breaks, Seep 70(*Overl we have ests tht he. thermopias
CCRISPRUCas jt cles oth uteri nd plaid DNA in viApplication No: [3842859
‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014
Page 29
GARNEAU tal Cie.
Te conpostion fain 4, wherein fe enzyme
metas city, deme activi, DNA reat,
DNA danage ay, deamination activity, dsmtae activity, alkylation
avy, depuration activity, xo activity, pyridine diner oning
activity, integrase activity, ransposae activity, recombinase activity,
polyaseatviy, igs acy, ease acy, pays ati or
tyes acy.
Claim St
The composition of in SY, wren te enya activism ay.
Chaim $32
The composton of cin wren females ey ntoduces a double
stan breakin the target DNA.
This endonuclease activity, which sms equ casS, i prot-yacer
spec and oration dependent), ee ap. 7c (scbing
experimen sling te ln ceavage avy of. thophias
CCRISPRIUCas system). See aso Abstract ("We also provide in vivo
evens ththe CRISPRICas system silly caves sid and
teceropag oul stand DNA wikia te poospce, at spec
sits”
Claim $6
The composition of cai 4, wherein te DNAapting RNA isa double
nol DNA argetng RNA ad th composition comprises oth rer
NA and an atvalor RNA, he dpe-fomning segment of which are
complementary and yb fom he soca segment ofthe DNA arin
RNA,
area ta cscosesompostons in which he DNAgting RNA,
compra tarete-RNA (cRNA) and an acivaor RNA (ie,
tracRNA), Such ompostions ar inherently present nS thermophis
cel,
Claim 64
A metodo sie spite motion of tt DNA, the method comping:
contacting the target DNA with:
Garant a. dsc etd fr ec modicaion ofa tt
DDNA eg, plaid or takriophge DNA) by contacting te get DNA
with DNA lrgeing RNA anda seed modifingplypeplie, Se
1p. 70 (*Overal, we have established thatthe S shermophilws CRISPRI/Cas
syle ces uth backronag and psi DNA in i, Ths
endoruckese act which senso eu as, pr spore pci
anita dependent”)
ng RNA, ora DNA poljuclvie nooing the sane, when
‘he DNALergeting RNA compris: 2 fit segment compring a uti
sequen thls eomplemenary oa sequen inthe taget DNA; and)
second segment that iat with site dicted mdilying polypeptide end
Garneau ot al. discloses DNA-trgting RNAS. See analysis fr claim 1,
above,Applicaton Nos 15'42,859
Third Paty Submision 37 CLR. § 1.290 filed Sepembe 5, 2014
Page 30
GARNEAU etal (Cite No.3)
(ijase- deed mollingplyepide, ora lyre neigh
sane, when th siete modifying plypeie conpi: (aan RNA-
binding prion tt intact wth te DNA-argting RNA and () an city
orion ht exis site-dree enzymatic avi
arcu tal. dso sites modifying polypeptides, Se amis
foci, ahve.
Claim 65
‘The method of claim 64, wherein the target DNA is extrachromosomal,
area ta. discoses target DNA Hat setchonesoral, ach as pNT
plasnid DNA, See, ep: “Squen of CRISPRL in te
afemestioned 30 clones iden eet spaces ($585) al of
‘hich were logs to pNT1 sequent (Tal 1 an Fig 1)?
Chaim 4
The method of cam wri ie nat activity mds axe
DNA.
Claim 75
Themetnd of clin 74, wherein he enzymatic city sunset,
netylransrase avy, demetylseatity, DNA reir atity, DNA
damage avy, deamination ati dimuaseatvity, alkylation ati,
¢epurinaion avy, oxidation atv, pyridine dnc foing atv,
negra att, resposee avy, eoombinase acviy,polymerse
activity, igase atv, belae avi, photolyase ato fesse
actly
Chain 16,
Themed of cain 75, ween te DNA-modiing ena acy
rucease activity.
‘Claim 77
‘Themetnd of claim 76, wherein he ules att intodresa due stand
bee inthe tet DNA,
areata. dso mtd in which he enzymatic Le, doable
stad ula ati modi te taget DNA, See usin faci
952.Application No: 13842859
Thi Paty Submision 37 CR, § 1.290 ied Selember 52014
Page 3]
Notice of Allowance and Fee(s) Due for 14/054 414 (U.S, Patent No, 8,697,359) (Cite No. 4
This publication discloses that atthe time of the earliest priority date of the US 201410068797 family, the *797 family
hhad not yet demonstrated that the CRISPR-Cas9 system could be used in eukaryotes, and it was not known whether such a system.
could function in cukarotic ool, Itwas not until the third provisional patent application in this family (US 61/757, 640 fled January
28, 2013) that it was demonstrated that the CRISPR-Cas9 system could be used in eukaryotes. See The examiner's statement of
reasons for allowance at page Sof Notice of Alloway.Applicaton Nos 15'42,859
Third Paty Submision 37 CLR. § 1.290 filed Sepembe 52014
Page 3?
MALLetal Cite No.8)
A geely modifi cell comprising econ se deed mdiing
poled comprising
Nal tl dls genetically nde cls comping eed
rudihng polypeptide, Special, Malet ses human 2957 cel,
human K5¢2 cel, and un inferences hat ae
sxnealy modified to crn amanmaln expressions
codpinized Ca) pron, Se aban pae'
“Sie diet mdithing polypeptide” essed in chi is defied in
rere [088 of US 204006879 i incude Cas).
an RNA ing prion ht inerats witha DNA-arting RNA; and
Malet al sds aside mollyigplypepie comping an RNA
biting potion tat inka witha DNA-arsng RNA, See page 823, co.
(rRNA fad oa romaly rns ened acsRNA is slice det
(pron osguenoe special cave target DNA sequences matching
thecRNA.
an att potion tates sitet enzymatic cv, when the
site of rzyatc acy is determined by the DNA-argting RNA,
Nal etl dies asie-eed odin polypepide comping en
acy shons site of emai aiviy is deterind by the DNA areing
RNA Seepage 823col | (rRNA fsedtoeoealytransnode
trarRNA isi det Cas) pron semen pel cave
tant DNA squnces mating the rRNA)
Claim 64
‘Ame ose pce modication oa tres DNA the mead
comprising contacting the taget DNA with
Nal tal dhs methods of sie pic modifi of get DNA wing
DNAargeing RNAS and seed modiyng olyepis. See abstract,
(@}aDNAtngetng RNA, ora DNA polynucleotide encoding these,
hein the DNA arating RNA compris 2) fist epmen comprising a
rule sequence tats complementary a sequent in th tget DNA;
and (h)a sco semen that inert Wiha ered modilyng
polypepiean
Malet al sos DNAargetng RNAS. See page 3, col 2 (Gilosng
asco RNA tareRNA ison transitions (feed 00s ue RNAS
(gRNAs)o dest C9 to cve sequences oie),
“DNA arting RNA” sth ter suse in cm 6s defn in pags
(0130) and (0136 of US 201410068797 to include “ide RNAS” hat can he @
single RNA molecule, such as @eeRNA-tarRNA fision
(isle modjingplypepide, ora pyri neigh
sane, hen the sieeked modilingplypepide compris (2) n RNA-
binding prion tt intact with te DNAartng RNA and (an city
Malet a cscs sie-dieted modifying popes. See age 23, col 2
¢ebegun by sytesizing« human coon-pinizd version of te Cas)
protein....").Application No: [3842859
‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014
Page 33
orion hl exis ste-dreed enymaticatiy “Siteiced modiyng polyepide” as used in claim 64s dling in
paragraph [O18] of US 2LAO6IT to include Cas,
Chaim 69
The metic 4, when ie eget DNA ipo omasoneing | Malet a divloes ste specicmniiaion of target DNA in uma 2937,
el
5¢2 an ined puriiet sem els, Se abstract
Claim 9
A met f promo sitespeife eavage and modification fated
DNA ina el, he mad comprising iran int th cel:
ale slows mets ofprmolig epic cave and
‘dtzaton of get DNA na cel itading DNA ugeting RNA and
sitet ming polypeptides. Se poe 82
(DNA aring RNA or DNA poymclenide enoning he sane,
herein te DNA-trgting RNA compris 2) fist segment compcinga
rl equnce tats complementary a sequen in th tet DNA;
and (a send erent interacts wih a ste iret modilying
polypepiead
Mali tal discloses DNA-‘targeting RNAs. See discussion for claim 64, above.
(ijase-dreed moliingplyepide, or polyrucleide ncding te
sane went sie-etd modiling polypeptide compris (0 RNA-
binding portion ta nets wih he DNAting RNA; and (6) an acviy
orion tht exis mule att tat crete stand breakin he
‘et DNA,
Malta dco ste ined modifhng polyenes. See dicusion oe
chim 8, oe
vn the sith dole stand reais detent the DNA reting
NA te conating ocr unde odin tat are pes or
nabomologous end joining or bmolog-ireted epi, andthe get DNA.
is cleaved and ined to produce adie’ DNA sun
alta los otaing under condos hata pemissve ir
onhonologns nd jiing or homolog dire air, esng in get
DNA being cleaved andthe ona produc a modified seen, See
page 24 col | an (dln geting of AVS! loin PPPIRIZC
ene hick hgh mers of so logue jinn (HE) vets
(cing deltos an inser wer observed upon texting wth Cos)
andgRNA),
‘Chaim 93
The meld of claim, he meth further comprising ontaing he eet
DN witha dnp, when th door pout, pation
fe dno polynuckde,copyo te dan poyrukeide, oa prion af
Nall lose method fre comping cotactng te rt DNA wi
donor polyuckide.Se page 824, col. 3 songs of doarApplicaton Nos 15'42,859
Third Paty Submission 37 CER. § 1.290 filed Sepembe 5, 2014
Page 34
MALLetal Cite No.8)
apy
one pleut no eat DNA
constuc oaitatebomologous eeuminaton (HR) meted integra),
Claim 94
‘The method of claim 92, wherein the method does at comprise contacting the
cell with door polyouckte,wrein terget DNA is modi such tat
ruck within th tg! DNA are dle
Nal tal dls: method tt do ot use door polymcketdeuch tat
‘nucleotides within the target DNA are deleted, See page 824, col, 2 (“AS
expected, NHEJ-mediated deletions for TI and T2 were centered around the
re sips)
Chaim 98
‘A mend of prodcing 2 genetically modified lina sje he thd
comprising
(Dodge:
when the DNA tating RNA compres fit segment
comprising amulet sequence that is complementary toa sequence in
the target DNA and
poled; an
the sane, wherein beste deed nodilig polypeptide comprises
RNA; and
(Banat prion that extiis ule avy tat casa
daxble strand breakin te ret DNA wher te st fhe double
strand re deterind hy the DN ging RNA the cnaing
ecu under oto thar permisine ornokoologous end
jn or holy repr, andthe txt DNA isla
antelope 2 malied DNA segue they rung
the ently mdifed cel; and
(a DNA retng RNA ora DNA polrackotie cog te sn,
(Wa seond eget at nrc wih ase deed odiing
(ia ied mong pol ppd oapolauceodeecoing
(@)an RNA Pinding pron a its with DNAagetng
Mata dcloses nvoduing DNA tugting RNAS and sit-reted
nila polypeptides ito 295, KS and ind pure! sr es,
Seecission clin 64nd, ahve
(U0) transplanting te geet nid el ino the sje.Application No: [3842859
‘Third Party Submission 37 C.E.R. § 1.290 filed September 5, 2014
Page 35
MALLetal Cite No.8)
Chim
The method of cain, the etd uber comprising conan tcl
with donor polyauclde, wherein the donor polnuceide, a prin ofthe
donor pojnucie,acoy of the dnp ora patina
copy ofthe donor yack integrin tet DNA,
Nall dls methods fuer ompring craig the cel witha
dan: polyrukide to bnegrde int the tg DNA, Session for
chin 3, hove
‘Claim 1h
The method of claim, wherein te metod oes at comprise conaingthe_| Natal. des method tt do ocr contacting te cl witha
cell wih donor oye, wire tert DNAismodied ach tat | donor pluck sta cote with there DNA are deed
ucleotides within the target DNA are deleted. ‘See discussioa for claim 54, above,
Claim 102
A method of ming target DNA ina genetically modified cell hat,
comprises
‘etd seuene enna an expen siete ming
polypeie hemetho comping ining inthe genetically tied
cella DNActgetig RNA, ora DNA polymuleotde encoding te same,
vii Nal tal loss methods of moding target DNA cls ictdcing
(ithe DNA targeting RNA comprises: 2) a first segment comprising a DNAargtng RNA and site-directed ming polypeptides. See discussion
rucetde sequence thas complementary toa sequence inthe target DNA; | far clims 38 and 0, above,
and (b) 2 sound segment tha interantswith a site ete modifying
polypepie; and
(ite site-directed modifying polypeptide comprises: 2) an RNAPinding
gorio tt inact wit the DNA-argeng RNA and () an acy potion
‘that exhibits mules activity
Dependent claims 40, 70, 95, 101, and 104 (omitted from the table) are al directed to lists of cells or organisms that
comprise eukaryotic cells and organisms. As disclosed in Cite No. 4 (Notice of Allowance for U.S. Application No. 141054,414),
demonstration of successful use of the CRISPR/Cas9 system in cukaryotic cells in the US 2014/0068797 family did not occur until theApplication No: 13842859
‘Thid Paty Submision 37 CER. § 1.90 filed September 52014
Page 36
filing ofthe thd provisional application (US 61/757,640; filed on January 28, 2013), which is after the January 3, 2013 online
publication date of Mali et al, Mali etal. discloses use of the CRISPR/Cas9 system human 2937 cells, human K562 cells, and human
induced pluripotent stem cells (see abstract,Applicaton Nos 15'42,859
Third Paty Submision 37 CER. § 1.290 filed Sepembe 5, 2014
Page 37
CONG etal (CiteNo.6)
Chaim 38
A geeicaly moe el womprsing «recombinant se deed modiing
poli comping
an RNA binding portion that interacts witha DNA
‘an tivity pion that existe Get emai, when te
sit enymatic avy is erin hy th DNA ging RNA
(Congetal dso gee modtied cel comprising seed
ding pole. Spey, Conta. dss nan and mse
cel tata geal nied o conn mannan expression sem
comprising odo ized Ca poi, Se abst an age 820 1
“Site diet ming polypeptide” essed in chi is defied in
razah [188 of the 97 publon inde Cas9,
Claim 64,
‘Amt of sie peri moist oes DNA the mead
comping contacting tert DNA wi
Conger al disses metodo st pce mieation of txt DNA wing
DNA areing RNAS and sie ied modiingpolypeptibs. Se asa.
{) aDNA-argtng RNA ora DNA polyno encding he sane,
‘wherein the DNA targeting RNA comprises (a first segment comprising a
ruck sequence tts complemen in th tet DNA;
and). seond see thinkers wih ase dete mdifing
ply and
(Conger al istses DNA ging RNAS. Sep. co 3 (longs
ofa cleric cRNA taerRNA hd fr programabe geno eitng
sig he CRISPRCs syste).
“DNA argting RNA” es wed in cm 6st in paragrps[013] and
[036] of US 201416797 to ine “ie RNAS at cn easing
RNA molecule, such as a crRNA-traerRNA fsion,
(iste ied modhng polyp, ora polyline the
sung, win este malin lypeptie cmp: RNA-
binding porn tht inteas wi th DNA-agtig RN ad) an ati
postin hat existed nea avi.
(Conger al isos sided modiing popes. Sep. 0, col. 1
(song codan-pinization of, pyogenes Cs fo exes in buman
293FT cel.
“Sided maifng pope” assed in claim sdf in
ere [188 of US 204006879 incu Cas.
rg DNA i pole ronan ine
in. Se pp. 89-20 nd ast pls, 2, 3.
Cngetal slo sitespcic modification of tut chrmosonal oi in
‘oun and mouse cel that ae geet vido comprise Cad
‘Amati of prong sie pes cvag ard ofc o tegt
DNA inaceh he meth comprising inducing ino thc:
Conta dilses methods of promoting siv-sf cleavage and
mdi fret DNA ina ely intrdaing DNA-apting RNA andApplicaton Nos 15'42,859
Third Paty Submision 37 CLR. § 1.290 filed Sepembe 52014
Page 38
CONG etal (CiteNo.6)
siete moihing polypeptides. Sep, &21-47
Asenind on pg 822, Candied ology iets HDR)
wa eset hE ow holy eel itu
‘pair of restriction sites, Cas9 and a mutant Cas9 nickase each catalyzed
ifegaton oft epi emp into EMT loos
Ascisusedon ug 82, th wildy Cad used aslo mie sit
speci double stand res, which cul be pied hohe on-
‘bomologeus ead joining (NHED) including deletions o insertions) or
bolo diced pi (HDR).
‘See also discussion fo aims 38 and 40, above
(i/aDNAcareng RNA ora DNA poljauetide ending he ane,
‘eine DNA argetng RNA compris (2) fist segment comprising a
rule sequence tats complementary a seqpece in th tget DNA;
and (ba seund een ht intra witha ste iret modtying
polpepie and
Cong etal discloses DNAarvting RNAS. See discussion fr claim 64,
above,
(ives eed modyng poppe, ora plyacide noting
sane, when the siete modi plypepide comps (2 RNA-
binding portion tt inert wih he DNAaretig RNA; an (6) an acviy
orion tt exis ucla tity tates dbl stand eck inti
tet DNA,
Cong etal dla steed malig plypepids. Se discus for
chim 8, oe
‘here ste th double stand rea deen hy he DNA-erting
RNA th coating ous under coniton tht ae pes for
rorhologus en ining holo repi,and th gt DNA
‘scleved and une o ruc a modified DNA sequen,
(Conger al disses contig unde condos iat ae prise for
onhoologs en jing or homology direc pair, skng i get
DNA bring clave anther jie i produce amid eno, Seep.
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