Haoyi Wang:
All right.
Thanks for having me here.
And it’s a really interesting meeting.
And so, I worked with Jackson Lab to establish
and improve the technology of using CRISPR
to make mouse model.
And my main lab, actually, is in Institute
of Zoology, Chinese Academy of Sciences in
Beijing.
In there we focus on making large animal models,
as well as therapeutic applications.
So, when we think about precision medicine,
or the topic of this conference, I think it’s
really -- we want to know is, by looking at
the genomic sequence of the -- of the different
patients, we can identify the genetic variants
that have something to do with the desistive
element.
Therefore, we can develop therapeutics for
them specifically.
And another layer of complexity is the epigenetic
modifications.
So -- and the misregulation of the genes.
So, for those two layers, we need better technologies
to model them in the organism, or in different
assays.
So, genome editing, obviously, is a very powerful
tool.
And those are the major tools.
And CRISPR-Cas, obviously, is the most powerful
one.
So, what they do is, basically, you can design
those protein to bind to very specific locus
in the genome and make a double strand break
that can be repaired by different repair pathway
that result in an allele.
Or you can put in pretty fine mutation there.
And the reason CRISPR is so powerful is different
from the other two.
You don’t have to design a new protein for
each locus.
You just design [unintelligible] that based
on the base pair ring of the RNA and the target
DNA.
You can guide the Cas9 that go to anyplace
to make the genome modification.
So, that case of work really accumulated to
this landmark paper that demonstrated you
can basically express those two RNA that recognizing
any DNA template and generate this double
strand break precisely.
And, followed quickly by those two papers,
showing optimization of the system can be
useful in the mammalian cells.
And what we did then is to show you can -- in
the mouse embryo and stem cells, you can simultaneously
knock off five genes with very high efficiency.
And that can be useful for making mouse -- cell
line models.
Also, by introducing the system into zygotes,
the enzymes start add in the genome -- or
the embryos.
Therefore, you generated a mouse with the
mutant in one generation.
And you can actually do this by: knockout
multiple genes at one step, as well as putting
in multiple specific permutations to different
genes in one step.
I want to point you to the supplemental data,
actually, which is -- we tried to knockout
that three.
And the knocking -- the genome editing is
very efficient.
So, this band is the mutant band, this is
the [unintelligible].
So, you can see all the embryos are kind of
mutated.
And we already see the phenotyping F0 animal.
So, when you inject them, day one -- in day
21 you see those pups showing the neonatal
lethality phenotype.
So, that means you can actually use this approach
to screen a large number of genes to see their
phenotype with a very small number of animals
in a very short amount of time.
And, in the following study, we showed, by
introducing different type of DNA template,
you can repair the break by introducing epitope
tags, or reporter genes, as well as loxP sites.
So, with all that set up, the system is comparing
to traditional gene carrying making mouse
model the cause is dramatically reduced.
And, more importantly, the time you saved
is most significant.
And, also, it’s very flexible.
You can theoretically work on any string you
want without have to do this first in good
MR stem cell strand and a stream algorithm.
So, still, the one bottleneck of this whole
procedure that does not allow high throughput,
is really the micro-injection process.
You need a very expensive setup, and also
very -- the most -- I think the most limiting
factor is really to find a very good micro-injectionist,
can do this very, very efficiently, and consistently.
So, to solve this issue, we developed this
zygote electroporation of nuclease technology.
So, basically, we treat the embryos as any
other cells.
We electroporate in the labs.
We just put in -- them into a cuvette -- electroporate
them in the commercially available electroporator.
And that allows you to deliver the CRISPR
agent uniformly to hundreds of embryos in
the same cuvette simultaneously.
And, after optimization, you can -- this is
one example, actually, is one of the rather
difficult locus we’re targeting.
So, here we are introducing two [unintelligible]
sites to this locus.
And so, if it’s targeted correctly with
the precise permutation you would digest in
this PCR product with both an -- either enzymes.
So, that’s exactly where you see -- so,
you can actually achieve a really a 90 percent
of [unintelligible] guiding our founder animal
by just electroporating them.
So, with that, I think we are really now -- can
do this with a high throughput manner.
You have one technician knows how to handle
embryos, they can quickly go through 20, 30,
50 projects in one -- in the -- in the -- in
the -- in one day.
And then, you can -- you can just transfer
them and do your phenotyping.
So, I think, with those we can quickly screen
candidate genes in founder animals that -- either
it’s from multiple genes in [unintelligible]
hit, or it’s many new genes you found from
exome sequencing.
And, also, you can generate human genetic
variant in the mouse ortholog by using a single
oligo as a template by electroporation.
So, in addition to genome editing making mouse
models, I think the Cas9 is also extremely
versatile protein.
You basically -- they have two independent
nucleus domains.
So, if you mutate either one of them, they
become a nick case.
They’re only nicking one strand of the DNA.
And, if you mutate both of them, they become
reprogrammable DNA binding protein.
They can guide it by RNA and binding anywhere
you want.
But it doesn’t cut.
So, by doing that you can actually fuse a
protein that have any function to do a very
specific -- almost anything to anywhere in
the genome.
So, we basically developed a system called
CRISPR-on.
So, basically, that’s a very simple idea.
You basically make -- fuse your dCas9 with
a very strong activator.
This is ten copy of the VP16.
And we show that you can actually bind to
a specific promoter, and activate the endogenous
gene very efficiently.
And you can activate three endogenous gene
simultaneously.
You can also control the reshow of their activation
by controlling the amount of RNA you’re
expressing.
And, also we showed, if you introduce a system
into one cell zygote, you can actually turn
around the reporter you co-injected in the
early embryos.
So, you can do this potentially in vivo.
But the system is still not perfect.
If you think about a complex disease, or a
complex transcription network, because if
now I want to activate certain genes and repress
certain genes, you cannot really do this in
the same cells because, if you co-express
those effectors, they don’t know which gene
they’re going to repress and which gene
they’re going to activate.
So, you have to have a mechanism to do this
in a multiplex way.
So, what we -- the solution we thought of
is this combination of this Cas9 protein and
Pumilio protein.
So, this protein is really interesting.
It’s an RNA binding protein.
So, it’s like a tail, if you’re familiar
with tail [unintelligible] technology.
It’s like a tail.
So, they have this repetitive domain, each
domain recognizing one nucleotide of the RNA.
So, now, you can do is you can -- any near
this domain to recognize the specific base
pair of the RNA.
Now, you can have this RNA base pair binding
sites linked to the end of the guide RNA.
So, now, you have a molecule that have the
-- so, okay.
So, the specific binding sites will recruit
a specific flavor of this PUF protein, fused
with a specific factor.
So, then, with this single RNA molecule, you
combined the information of their function
and their target on the same molecule.
So, now you can do this with multiplexed manipulation.
So the first thing we tried is, by just simply
adding up to 47 copy of the binding site,
eight base pair binding sites, that doesn’t
interfere with the CRISPR system.
So, this is not fused, and this are fused.
With different copy number they perform equally
well.
And then, the real experiment is we express
the system together with different flavor,
different PUF protein, and fused with the
VP64.
And we express, say, the PUFa with four different
-- with the guide RNA fused with four different
binding sites.
And they only activate when you express the
guide RNA that have the PUFa binding sites.
So, that means it doesn’t activate in other
sequences.
That means the system is very specific, and
they can work independently with each other.
So, we showed go ahead, and this system can
be used to activate the endogenous gene.
And, because we are now recruiting many copy
of effector -- not just one copy by direct
fusion, you can activate the genes much more
efficiently.
So, this is the -- this is the Casilio activation.
This is direct fusion activation.
So, this in OCT4, and SOX2.
More importantly, the experiments do show
you can simultaneously activate in one gene
and dilating in the other gene.
So, that’s basically proved the concept
that you can actually have this information.
The PUFa was recruited to the gene they want
to activate because the PUFa fused with the
activator, and the PUFc I think we’re talking
whatever -- recruited to the gene they want
to repress.
And with the PUFb protein fused with the repressor.
And, more than activating the promoter, which
showed you can -- the system can recruiting
[unintelligible] into the enhancer, and therefore
manipulating the gene expression from binding
to the enhancer, and modify the histo.
So, here we showed you can use the Casilio
system to binding to the enhancer of the OCT4.
And that actually, interestingly, activating
the gene more efficiently than the direct
fusion of the dCas9 and the [unintelligible]
transfer is fusion.
And one last thing is really -- I think it’s
very interesting to study the chromosome structure,
because that’s another layer of epigenetic
regulation.
And here we show, because you can recruiting
many protein to the one locus you can get
better signal to noise when you label a specific
genomic locus.
So, we showed that when you use more copy
number of the binding sites, you get a better
labeling of the telomere when you use a GFP
to label it.
And this is a quantification showing where
you use more copies you get more signals.
And, also, the -- a better number of foci
being identified that’s closer to you would
predict.
And, by co-staining, we show the labeling
is specific.
It’s co-localized with telomere associated
protein.
And the same thing can be shown for labeling
centromere.
And, obviously, a system can work simultaneously.
So, you can co-label two genomic loci with
two different colors simultaneously.
And, because this [unintelligible] feature,
we can now use much small number of guide
RNA to label unique sequence.
I think that’s a goal.
And we do observe a very nice labeling of
unique sequences.
But they still need to be fine-tuned to really
work well.
So, with this platform, I think it’s very
versatile.
You can use this to do multiple X function.
And directly multiple gene, you can recruit
multiple protein to specific locus, do labeling,
or do other function.
And the other thing we want to explore is
to really use to recruit a complex that can
work in a synergetic way.
So, with that, I think we can potentially,
in the future, to model epigenetic abnormality,
as well as gene regulation networks.
So, one last thing I want to -- I want to
raise -- just, it’s a question really.
Because we think about from the bench to the
bed side, other than diagnostics, I think
a very important thing is therapeutics, especially
for a lot of those [unintelligible] disease.
We have strong, strong evidence of this genetic
-- these gene is a causal gene, and its mutation
causing disease.
So, now we have the tool to really go there
and correct that endogenous gene.
That would be the, I think -- the fundamental
solution of those disease.
And should we actually think about identify
a list of actionable disease and genes that
is really -- is really bad, so when you see
this in the small kid you want to act on it
right away.
And will there be a mechanism we can develop
to a relatively cost effective model for the
therapeutic development?
Because each patient most likely going to
be unique.
They’re going to have different -- maybe
the same gene, but different mutation.
How do you develop a therapeutic approach
that can cure most of the patients?
And, whether we will have a mechanism to faster
approve those therapeutics for some of really
disease, I think it’s -- you know, we have
the tools, people are dying.
I think people should work on it.
And, also, how do we evaluate the risk and
benefit?
Because, when you do genome editing, I think
nobody can assure there’s no off target
whatsoever.
Nobody can ever be sure you don’t make any
other genetic mutation.
So, if it’s -- if the modified cells’
getting into our body for decades, then there
is a chance to develop cancer from it.
How do you know it is not from genome editing,
but from some other [unintelligible].
So, I think -- I think it’s really dependent
on the disease and how devastating that is.
But, this is something to be considered.
And, even one step further is how do we define
the norm of the human genome and what is normal
human, what is normal genome?
And where we draw the line to make that modification?
If the risk allele is worth editing or not,
and do we go to germ land at all?
I think that’s an interesting question,
and it becomes a reality I think people should
think about it.
So, with that, I’d thank a lot of people
involved in this for really -- it’s a great
collaboration within JAX with the GET group.
There are a lot of key people contributed
to developing the thing, and improving it,
as well as the reproductive science group.
And my lab there.
And also, I have a setup in Beijing that I’d
like to thank my director there, and the fundings.
All right.
Thank you.
Howard McLeod:
Thank you very much.
Questions?
Once you’ve picked your job -- Dan.
Dan Roden:
With this -- the first technology you can
overexpress -- you can really drive up expression
of any gene to sort of massive levels in a
-- in a -- so, does -- is that -- is -- I
don’t usually think about increasing gene
expression by 20,000 fold, which is what you
can do with this technology.
Is that --
Haoyi Wang:
I think artifact, because it really depends
on the basal level of the gene, right?
Because it’s not -- if it’s it’s zero
then you can evenly increase it.
Dan Roden:
Right.
Haoyi Wang:
So -- right.
Dan Roden:
So, my question is, is -- have you been able
to think about or see deleterious effects
of that kind of overexpression in -- across
cell lines or across genes?
We struggle -- we struggle with the idea of,
you know, what would happen if you increased
the number of sodium channels in a heart cell,
not one fold or two fold, but 25 fold?
Would that be a bad thing or not?
And can a cell -- would a cell ever do that?
Haoyi Wang:
I think that’s really interesting.
I think there’s so much unknown there.
And, also, think about the basal forms.
We some very interesting thing about when
you activate the gene a few hundred fold,
an aciform totally gets skewed.
Right?
And there is all unexplored.
And I think it’s very interesting to look
at.
Dan Roden:
Okay.
So, no answer.
Haoyi Wang:
But it -- like we activated all four for hundreds
of fold in T-cells.
And that does have activating other genes,
unrelated.
I think it’s through the secondary fact.
Howard McLeod:
Bob.
Robert Wildin:
So, I’ve -- two -- one question and one
comment.
So, the imaging that you did of the nuclei
-- is that an -- in an amplified target situation,
or could you use that to visualize the presence
or absence of a single nucleotide change?
I mean, could it be a fish for single nuclei
change?
Haoyi Wang:
I don’t think so, because right now what
we showed -- most of the data are in telomere,
or centromere.
There are still a lot of repeats.
Robert Wildin:
Okay.
Haoyi Wang:
And the single -- the unique sequence -- you
still need more than five guide RNA to tell
it.
Robert Wildin:
Okay.
Haoyi Wang:
So, I think we’re not there yet to show
that type of revolution.
Robert Wildin:
Okay.
And, in terms of potential catastrophic disease
targets, I would suggest IPEX, immunodysregulation
polyendocrinopathy enteropathy X-linked, which
babies are born with a severe autoimmune disease.
There is a treatment, which is bone marrow
transplant, but they’re often not well enough
to get the transplant initially.
Haoyi Wang:
Yeah.
Robert Wildin:
And they have a very difficult course, initially.
And one could think about trying to use this
to correct a very small fraction of T-cells,
which is all that you need, actually, to reverse
the autoimmune disease.
Haoyi Wang:
Yes.
We’re actually doing a lot of work in the
T-cells, and [unintelligible] cells.
So, I’ll write down the name of this after.
Howard McLeod:
Any other specific questions?
[end of transcript]
