Critique of Nicole Waynes Grant Proposal

Abstract:

Strengths:

Good big picture view of epigenetics and its importance in brain development
Clear statement of aims and organized progression of ideas.

Weaknesses:

It seems a little disjointed after you mention Grin2c then jump into There is a specific H3K27
demethylase. What is the connection between this demethylase and Grin2c? Maybe state your
hypothesis here and have more of a transition.
Explain what the dcas9-Kdm6b fusion protein targeted to the Grin2c promoter region would do?
Are you trying to knock in Kdm6b? Knock down? Its still a bit vague how you will use CRISPR to
test effective demethylation.
Also when you say you will demethylate H3K27me3 at the Grin2c promoter I think that you
want to make it clear you are demethylating the histone/protein not the promoter (DNA). Or if
you are referring to H3K27me3 as a nucleosome (both protein+DNA) that should be clear as
well.

Specific Aims:

Strengths:

Clear statement of goals and hypotheses

Good introduction into the key molecular actors

Weaknesses:

How will you determine its role more broadly in inducing gene expression programs during
development? Seems like a pretty broad statement.
Maybe you could reword the title of Aim 2 since its quite long winded and broad. It seems to
me that in Aim 1 you are testing for sufficiency of H3K27me3 in grin2c expression and aim 2 is
testing the sufficiency of Kdm6b in demethylation and/or also grin2c expression. So just say
that.

Public Health Summary:

Strengths:

Clear explanation of the importance of Kdm6b in the cell

Autism gives a pretty compelling health target

Weaknesses:

It seems that a lot of the project is focused on Grin2c but there is no statement about why it is
important to study Grin2c specifically. What is Grin2cs role in development? What will happen
if it is continually repressed and never transcribed?
Significance:

Strengths:

I like the organization of your ideas

More of the significance of Grin2c that is mentioned here

Weaknesses:

Maybe link more of the importance of epigenetic modification+histon demethylation back to a

neurological disease like autism. Gives more a sense of urgency.
When you say loss of kdm6b leads to unsuccessful neurogenesis, please explain how it is
unsuccessful. Did the neurons just die? Were they not differentiating? Differentiating in wrong
patterns?
o You could tie this to a neurological disorder which would further bolster your public
health/significance

Innovation:

Strengths:

Good intro on how CRISPR is useful and how it works

Weaknesses:

Now that youve explained what CRISPR is in sufficient detail, maybe you could expand on how
your work is novel.
o Is there a particularly novel way you are using CRISPR?
o What has been known so far and how is what you are doing in your research new and
exciting?
o You could also talk about the advantages of using CRISPR over some older techniques
for editing the epigenome

Approach:

Strengths:

Very thorough description of research methods and expected action in the cell
Good explanation of the control using GAPDH in RT-qPCR
Your schematic with the 3 plates is very clear

Weaknesses:

You mention that Kdm6b will ideally demethylate this promoter. How will you validate that
demethylation has actually occurred? Is there some sort of reporter for demethylation? This is
critical because if you were interpreting your results and didnt see a difference in Grin2c mRNA
levels how would you know if it was conclusion 1 that demethylation doesnt lead to Grin2c
expression or conclusion 2 that no demethylation had even occurred?
Overall: Good work on this grant proposal! Very detailed and thorough treatment of the topic. I just had
some minor suggestions for clarifying some of your language and making sure ideas connected. Good
luck!