Hello, everyone, and welcome
to this Integrated DNA
Technologies webinar
on CRISPR genome
editing with a
high-fidelity Cas9 enzyme.
My name is Dr. Hans
Packer, and I'll
be serving as the moderator
for today's presentation.
The presentation
today will be given
by Dr. Christopher Vakulskas.
And Dr. Vakulskas
is a staff scientist
here in the Molecular
Genetics Group
at IDT, where he works
on new enzyme development
and purification methods,
including the work that
led to the Alt-R S.p.
HiFi Cas9 nuclease.
Prior to joining
IDT, Dr. Vakulskas
was here in his PhD at
the University of Iowa,
where he studied genetic
regulatory circuits
in pathogenic bacterial species.
And then he went from there
to being an NIH postdoctoral
fellow at the University
of Florida, where
he studied RNA binding proteins
and transcriptional gene
regulation.
Dr. Vakulskas'
presentation today
should last about 35 minutes,
and following that presentation
we will answer as many questions
from attendees as possible.
And you can ask your
questions anytime
during or after
the presentation.
The best way to do
that is to type them
into the Questions box, which
you'll find in the GoToWebinar
Control Panel.
And that's at the right-hand
side of your screen.
And if you see, there's
a little up-arrow symbol
that you can click
that, and that'll
pop that window out
and make it larger.
It makes it easier to type
your question into there.
So yeah, that's how
you'll communicate with us
during the presentation.
And then at the end
of the presentation,
I will read as many of those
as possible to Dr. Vakulskas,
and we'll have a
conversation about the topics
that you're interested in.
Also, in case you need
to leave early today
or you want to revisit
this webinar later,
share it with someone, we
post all of our webinars
on our Vimeo and
YouTube channels.
We also have a video library
on our website, idtdna.com.
And if you look under our
Support and Education stuff,
you'll find our video
library as well.
So these are the links
to our video libraries.
We'll be sharing the link
to this webinar, also,
by email with you after
the presentation--
in a couple of days.
And then, we also get
asked about slides.
We post the slide
decks for our webinars
at our SlideShare site
shown on the screen,
and we will actually
have that uploaded today.
So you'll get a
link for that today.
Someone is working
on that right now.
And at any of these
resources, you can go look,
and you'll see we have
plenty of CRISPR webinars,
unrelated topics that kind of--
how to use our system, how
CRISPR genome editing works.
So there's a lot
of great content
already there, plus
NGS, qPCR, genotyping.
We do a lot of these.
So I think that's it for
my housekeeping stuff.
And with that, I'm going to
change the presenter over
to Dr. Vakulskas so
he can get started.
All right.
All right, thanks for that
nice introduction, Hans.
And so I'm Chris Vakulskas.
I'm a staff scientist at IDT.
I'm going to talk to you
today about a project we've
been working on for the
better part of two years--
trying to develop
a Cas9 mutant that
reduces off-target effects.
And just to give you
an outline of what
I'll be speaking
about today, I'm
going to go through some of
the basic background on CRISPR,
talk about the products that
IDT sells, how it works.
And then move on to
what I think is going
to be the main topic
today, and that
is the notion of
off-target editing.
And really, what I'm
going to hit on the most
is that the delivery
mechanism matters.
And by that, I mean, how
you're getting the Cas9 protein
into the cells-- whether
you're producing it
from a plasmid, from an mRNA,
or producing the protein,
purifying it, and
adding it directly.
And then, also, how you're
getting the guide RNAs
into the cell, whether you're
producing them off a plasmid,
lentiviral, or synthesizing
them and adding them directly.
So delivery mechanism matters.
And then I'm going to move
on to talk about a couple
high-fidelity Cas9 mutants
that have produced and reported
in the literature.
These were mutants
that were made
for plasma delivery of both
Cas9 and the guide RNAs,
and I'm going to show
you some data that
says that they don't work
as well with RNP delivery--
or ribonucleoprotein.
And the general
trend seems to be
a reduction in
off-target editing
at the expense of
on-target potency.
And then finally,
I'll go on to talk
about the rationale
behind why we decided
to make our own mutant, the
bacterial screening strategy
with which we developed it.
And finally, go into
the data describing
how well it works on target
compared to the wild type Cas9,
as well as these other
literature Cas9 proteins,
via RNP delivery.
And then, of
course, a discussion
of how well it works to
reduce the off-target effects,
and then go on to some
real-world applications
of this protein.
So we've been sending this to
beta testers for quite a while
now, as well as doing a
lot of in-house testing.
So I'll show some editing
experiments in primary cells.
Give you a hint of what it
can do in other species.
And finally, go
on to what I think
is, ultimately,
what a lot of people
are going to do with this,
and that is facilitate
homology-directed repair.
So just to get into the
basics of CRISPR, at IDT,
we sell two different
Alt-R CRISPR products.
The Cas9 from Streptococcus
pyogenes as well as
the Cpf1 protein from
Acidaminococcus species.
Cas9, of course, recognizes
the canonical NGG PAM.
So anywhere in the genome where
you've got a DIG nucleotide,
you could theoretically
have a Cas9 cleavage site.
Cpf1 recognizes the TTTV--
or triple T followed by
anything other than another T--
PAM site.
Cas9 can leave blunt
overhangs, where
Cpf1 leaves 5 prime overhangs.
And essentially,
all you're doing
is using these RNA
guided DNA endonuclease
to make double-stranded
breaks in live cells.
And towards this end, you
can either do it to disrupt
a gene's function by allowing
the native, non-homologous,
end joining pathway to repair--
ultimately resulting in
some sort of small insertion
or deletion, otherwise
known as an indel.
And alternatively,
you can replace
whatever it is that you're
cleaving with homology-directed
repair, as long as you
include a donor DNA,
whether that's in the form
of a single-stranded oligo
or a double-stranded fragment.
So we're selling
the Alt-R system,
and this is a two-part
RNA system for Cas9.
And so the Cas9
will be the majority
of what I talk about today.
And in this two-part
system, this closely
reflects what actually
happens in the bacterial cells
in the primitive immune system.
Where you've got a targeting
CRISPR RNA, or crRNA.
In this case, we are selling
a 36-mer with 20 nucleotides
of target sequence, and 16
nucleotides that hybridize
to the universal tracer.
And this 67-mer universal
tracer RNA, of course,
hybridizes back to
the targeting RNA,
and also interacts
with the Cas9 protein.
And it's also true that
people have appended these two
RNAs to form a single
guide RNA, and I'm
going to show you some of the
benefits and disadvantages
of either system.
So at the bottom, here,
on the left-hand side,
you can see the
two-part system where
you've got this 36-mer
targeting crRNA hybridized
to the universal tracer.
And then the single guide
form, where you just
got this loop here, and
the full RNA molecule.
So for the two-part system--
these are not ideal to express
in the cells from plasmid DNA.
You would need to add on
transcription terminators,
things like that.
For the same reason, not ideal
for in-vitro transcription,
or, I guess, IVTs.
We are an oligo manufacturer.
They're very efficient to
make with chemical synthesis.
And we've been able to
truncate them and make
them a good deal shorter,
and still maintain-- or even,
in some cases, increase potency.
So for the crRNA,
we've got this 36-mer.
And since it's so short, we
can make it on the cheap.
The 67-mer tracer-- we can
make it cheaply, as well.
And because we're making
them in a chemical fashion,
we can modify, chemically, any
position we want to do things
like allow for escape
of the immune response.
Whereas with these
single guide RNAs,
these are ideal for DNA
expression cassettes.
You can put a
terminator on them.
They're going to
be made in-vivo.
For the same reason,
they're ideal to be
made from an in-vitro
transcription reaction.
That's relatively low-cost.
We find they're inefficient
for chemical synthesis,
mostly due to the
length-- they're longer--
and then also, the higher cost.
And then if you're
making them chemically,
it's costly to do this.
And if you're making them
with in-vitro transcription
reactions, it's very,
very difficult to modify,
especially the
internal nucleotides.
So for that reason, we do
favor the two-part system.
And we also favor
it because we see
a reduction in
toxicity and induction
of the innate immune response.
So this is just an example--
an experiment, here,
where we're showing you
a HEK-293 immortalized
stable cell line that
constitutively expresses Cas9.
You can see the cells
look quite healthy.
If we take an sgRNA
that's produced
from an in-vitro
transcription reaction,
you can see a marked
amount of cell death.
And if we do the same experiment
but use the two-part system,
you can see the cells are
mostly indistinguishable
from the negative control.
So we also see
evidence for induction
of the immune response.
These are qPCR
reactions, and we're
looking at one specific
marker for the induction
of the immune system.
And on the left-hand
side here, you're
seeing a human normalizer gene.
So we expect, and in fact,
we see everything is equal.
This is the IVT sgRNAs--
the two-part and the
cells only are all right
on top of one another.
And then for the actual
immune system marker,
you're seeing the
two-part system,
and the cells' only
negative control
are on top of one another.
Whereas the sgRNA IVTs,
you see this huge CQ shift,
indicating a large induction
of the immune response.
And this is also true--
we've tested this with a lot
of other immune system markers
as well, and we just don't see
this with the two-part system.
Nevertheless, you can
implement CRISPR-Cas9 cleavage
in a variety of different ways.
You can supply the Cas9 protein
either as a purified component,
adding it directly to
the guide RNA complex
and then transfecting
the resulting RNP.
You can supply Cas9 as part
of the stable cell line
with the Cas9 gene
integrated in the genome.
You can use in-vitro
transcription
to produce a Cas9 mRNA.
And alternatively, you
can use either a plasmid
or a lentiviral delivery
system to supply Cas9.
And it's basically
the same story
with the guide RNA complex.
You can use our Alt-R
two-part system.
You can use the
aforementioned sgRNA.
You can also produce an
sgRNA by transfecting
a double-stranded DNA
fragment that encodes them.
Or alternatively, as
for the Cas9 protein,
you can use a plasmid
or lentivirus.
And you can mix and match
these however you want.
And we, of, course favor
the two-part Alt-R system.
Recommend it with RNP delivery.
And really, it
couldn't be simpler.
And in fact, all you do is
order your targeting crRNA,
mix it into 1 to 1 molar ratio
with the universal tracer.
You hybridize it by heating
to 95 degrees for 5 minutes
and allowing a slow
cooling on the bench top.
And a few minutes
after cooling, you
can add the guide RNA
complex from this reaction
to purified Cas9 protein--
again, at a 1 to 1 ratio.
Let that form in
RNP for 10 minutes,
and then the
resulting complex can
be delivered by whatever your
favorite delivery mechanism is.
So we like this system.
It's very, very easy to do.
You escape the immune system.
You don't have as much
toxicity, but there's also
another reason we like it.
And this all hovers
around the idea
that the delivery
mechanism matters,
and most prominently
for the reduction
of off-target effects.
So Cas9 from pyogenes
is known to produce
these off-target effects.
And what I've got
on the screen here
is an example of
the GUIDE-seq work
from Keith Joung's lab at Mass.
General.
And what they show is
that if they include
in their CRISPR-Cas9
editing experiments--
along with this EMX1 guide--
a double-stranded DNA
fragment and allow
that to integrate at all
the double-stranded breaks
with non-homologous
end joining, they're
able to sequence and find all
of the double-stranded DNA
breaks that were in the cell.
And they see, of course,
that this on-target site--
the intended edited
site for EMX1--
is the most
frequently-edited site.
But they also find all of
these other off-target sites,
some of which have a relatively
high frequency of editing.
And you can see,
as expected, these
are very similar to the
intended on-target site,
with a few changes.
And what we've
found in-house is--
the extent of
off-target editing seems
to correlate with the delivery
mechanism of both the guide RNA
complex, as well as Cas9.
And it seems to be
worse when you've
got plasmid-expressed
guide RNA and Cas9.
And I think I could
go on and on and try
to advertise for
the system we sell,
but I think, ultimately, people
are going to believe this
if they see it not
from just our group,
but from our friends
at Thermo Fisher.
And what they've shown in
this very nice experiment
is a western blot where
they show you where Cas9 is
and how long it persists
as a function of time,
and also as a function
of delivery mechanism.
And they correlate
that with detecting
levels of off-target editing.
And what they show
here is with DNA
plasmid-based Cas9
expression, it actually
takes a few hours to get Cas9
present to its steady state
level in the cell.
And then it's also stable
outwards to 72 hours.
So it comes on pretty
strong after some time
and is not easily gotten rid of.
With mRNA, you seem to see
Cas9 produced a little sooner,
but it also seems to be very
stable as a function of time.
And then if you purify Cas9--
form a guide RNA
complex in a test tube
and transfect that directly--
you can see that you'd
get a lot of Cas9 protein
that's present very
early, and that that
is either degraded or
deluded by growth over time
and is almost gone by 72 hours.
And what I think is so cool
about this is if you correlate
that with off-target editing,
you can see that in black,
here, with the plasmid DNA--
these two known off-target
sites--
you get the most
off-target editing
with plasmid delivery
when Cas9 is most abundant
and hangs around
for a long time.
It's an intermediate
effect when you're looking
at mRNA, which makes sense.
And then if you look at protein,
where Cas9 does not hang around
as long, you see the lowest
levels of off-target editing.
And we essentially
see the same thing
when we do these
experiments here at IDT.
And what I'm comparing here is
low-level constant expression
of Cas9 in this HEK-293
stable cell line,
and I'm comparing that to
RNP-- or ribonucleoprotein--
delivery of Cas9 in
this same EMX1 guide.
So what you're
looking at here is--
in dark blue is the intended
on-target site indel
formation at that site.
In light blue is the top known
off-target site for this guide.
And in gray is the second known
off-target site for this guide.
And simply switching
the delivery mechanism
from stable cell
line to RNP, you
maintain the same level
of on-target editing.
But even at the highest
dose, you dramatically
reduce off-target editing.
And then, what's really
interesting about this is you
can dilute that
complex even further--
for the most part,
maintain on-target editing
until you get to this
lowest concentration.
But you can also reduce
off-target editing
even further.
So this all hits back
on the same notion
that the delivery mechanism
does, indeed, matter.
And if reducing off-target
edits is your goal, using RNP
can get you a part
of the way there.
And as you might imagine--
in a perfect world,
we'd be able to predict what
the best guide is going to be,
which ones are going to have
the fewest known off-target
effects, and choose
those guides and not
have to worry about
any of this stuff.
And in fact, there have been
several very good attempts
at making prediction
algorithms to determine
whether guides are good or bad.
And what we found is that
it's just very, very difficult
to accurately predict
Cas9 off-target sites.
And I think this has a
lot to do with there just
being a lot of things
that we don't yet
understand about
cleavage preferences
for the Cas9 enzyme.
And I think that basic
concept is best shown
in this experiment.
So we've taken a cell-free
Cas9 cleavage experiment here.
This is very similar to
digenome-seq or SITE-seq.
It's a proprietary
system we have in-house.
We're using this
androgen receptor guide.
And if you look at
the guide sequence,
there's nothing
obvious about this
that would say-- it doesn't seem
to be a horribly repetitive.
There's nothing obvious
about it that says,
this is going to be a bad guide.
And what we've
done here is we've
shown all the cleavage products
from the cell-free experiment
and put them in rank order.
I've colored the on-target
cleavage in red here,
and thankfully that is the
most frequently-edited site.
But you can see all these
different off-target sites
in blue and green.
And what is the most scary
thing about this diagram
is that I've colored in green
the ones that are predicted
by the SVM algorithms,
and it's less than 10%
of the total off-target editing.
And I think that this is
certainly a worst-case scenario
for off-target editing, in
that there's no transcription
factors.
There's no chromatin around,
since this is purified DNA.
So Cas9 is more
free to do editing.
So again, this is a
worst-case scenario.
And also, it's going
to hang around forever.
You're not going to get
degradation of the RNP complex.
It's not going to get
dilution by growth.
But even considering that
these most-frequently edited
off-target sites
were not predicted
by the off-target algorithm.
So I think if you're
in a position where
off-target editing is a
concern, this is a scary graph.
And there have been
a number of attempts
to reduce off-target effects.
And obviously, we found that
delivery of Cas9 and the guide
RNAs [INAUDIBLE] and RNP really
gets you part of the way,
but it's not a total solution.
It doesn't eliminate
off-target editing.
And we've also tested, in-house,
some of these other mechanisms
to reduce off-target editing,
like reducing the length
of the targeting crRNA.
So normally we use
a 20-mer, and there
have been some
literature reports
of shortening that to 19,
18, or even 17 nucleotides.
And we find that
that has a mixed bag.
We find that for some
guides, it does, indeed,
reduce off-target
editing and you still
get good on-target editing.
But it's really unpredictable.
In a lot of the
guides that we tested,
you see a marked reduction
in on-target editing.
And that sort of renders
it not a good option.
And I think it's the same thing
for chemical modification.
So we've tried to introduce
specific chemical modifications
at different points within
the targeting crRNA.
And sometimes we see a
reduction in off-target editing,
but oftentimes, that
correlates with a reduction
in on-target editing.
And so that leads us to
the last thing on my list,
and that is the so-called
high-fidelity Cas9 mutants.
And at the time we
started doing this work,
throughout most of
the development,
there were only two of them.
There's now actually a third.
But the two proteins
that were engineered
were the eSpCas9 protein
from [INAUDIBLE] lab
at the Broad Institute,
and then this SpCas9-HF1
from Keith Joung's lab.
And these were a really
fantastic and clever way
to address this problem where
they looked at the crystal
structure of Cas9 in
complex with the RNA and DNA
target site, and made
pointed mutations to reduce
the affinity for either the
targeting or non-targeting
strand of DNA.
And I think the idea
was to eliminate as many
of the off-target, weaker
interactions as possible
and still preserve
on-target effects.
And in the papers,
these were all
done with plasmid delivery
of Cas9 in the guides,
and they seemed to work very,
very well in both cases.
And before we got
into the business
of trying to make
our own proteins,
we thought that we
would test both of these
and find out if they worked
as well with RNP delivery,
because that's our
favorite way to do things.
So we introduced
the amino acids that
comprised these particular
mutants in the context
of our Alt-R S.p.
Cas9 protein, purified
them to homogeneity
by the same process as
the commercial material
we sell, and then
we tested them.
To start off with, to look
at on-target performance
comparing-- and I
think we've looked
at greater than 50 crRNAs and
greater than 10 genomic loci
at this point.
And in the experiments
I'm going to show you,
we've tested these
comprehensively,
looking at immortalized
cells with lipofection.
And then also, T7E1 is
the readout for this.
But keep in mind
we've also repeated
a lot of these experiments
with both electroporation,
and also using NGS as a readout.
And what we found
from these experiments
is actually quite striking.
And
So on top here is our
two-part Alt-R system.
Everything it delivers is 10
nanometer RNP by lipofection.
These are 12 guides
within the HPRT locus.
And again, these are in
immortalized cell lines.
What we find is that the wild
type protein, for the most
part, seems to
function pretty well
with a majority of these
guides, save for a few.
And the eSpCas9 protein seems
to work only at about 50%
of sites, taking a
massive hit at some
of these latter HPRT
sites and a few in here.
And the SpCas9-HF1-- again,
when delivered as RNP,
seems to only work at a
fraction of these sites.
And again, I don't think that's
a failing of these proteins so
much as just a statement
of how they were made.
They were made for
plasmid delivery Cas9,
and that is, indeed,
how they work.
And just to show that
this is a universal thing
and not limited to
the product that we
sell, our good
collaborators at Stanford--
Danny Dever and Matt Porteus--
also put these into
human primary cells
instead of immortalized cells.
And they used
chemically-modified sgRNAs
instead of the two-part system,
and also electroporation
and analysis by NGS, and they
show basically the same story.
For the majority of these
clinically-relevant loci,
the wild type protein
works pretty well.
And there are some significant
hits in on-target editing
with both of these proteins.
And at the time,
we really wanted
to pick the best of these and
hopefully license and make
a commercial product.
And what we found is that
they didn't work well as RNP.
And in hindsight,
this makes sense.
They were made for
plasmid where you
get continued and
long-lasting Cas9 synthesis.
And we didn't want
to use plasmids,
because they're prone to
toxicity and immune system
stimulation.
So none of them worked well.
Reduced off-target editing
occurred at the expense
of on-target function.
And my background is that
of a bacterial geneticist.
I've done a lot of
bacterial screens,
transposon [? hunts, ?]
directed evolution.
And I thought that, OK, maybe
I can solve this problem
by doubly-selecting
for mutants that
avoided off-target editing,
but also were able to maintain
on-target potency.
And the bacterial system we used
is simply a two plasmid system,
here, that I've got
pictured in this cartoon.
And in the first plasmid,
you have an on-target site
that you must cleave
to avoid the expression
of a bacterial toxin
that's present.
So you have to cleave this
plasmid if you are to survive.
And on the second plasmid,
you have an off-target site--
a known problematic off-target
site for this guide--
and an essential gene that
the organism needs to survive.
So you have to avoid
cleaving this plasmid,
and successfully
cleave this plasma.
So we made a library of Cas9
mutants with low-fidelity PCR.
We screened about a quarter
of a million of them.
We got roughly 100
hits from the screen,
and we put it
through the wringer--
changing on and off
target site pairs,
doing repeats, et cetera,
et cetera-- and ultimately,
purifying and testing
the best of these.
And this is the
data that came out
of that original primary screen.
And this is, again,
rank-ordered by what I'm
calling a discrimination ratio.
So that's the ratio
of on-to-off target
editing for this
particular guide sequence.
So the higher the number, the
better the particular mutant
discriminates on
from off target.
And these sites were picked
because wild type does not
actually do a good
job of discriminating
at this particular guide.
And you can see,
out of this screen,
we were able to select a
handful of these that actually
had a phenomenal increase in
this discrimination ratio,
and several others that
were middling effects.
But we tested the best of these.
Purified a handful of them.
Tried different combinations.
And we ultimately settled on one
particular attractive mutant.
And we tested it using the
same sort of experiments
that we examined the original
high-fidelity proteins with,
And we did this,
again-- lipofection
at the HPRT locus with
the RNAiMAX region
from Thermo Fisher in
immortalized cell lines.
And also, our good collaborators
tested with electroporation
into--
well, that's not right--
into primary cells.
And what we found
was really striking.
And that is that our Alt-R
HiFi protein-- our best mutant,
here, in orange--
functioned the same as
wild type at the majority
of these HPRT sites,
save for a handful.
And if we look at
the same experiment,
but delivered with
chemically-modified sgRNAs
delivered with electroporation
into primary cells
by our collaborators, we see
it's basically the same story.
Our protein is largely
indistinguishable
from the wild type.
And if you're going to show that
you've made this protein that
reduces off-target
effects, I think
you have to actually show that
it reduces off-target effects.
So what I've got here
in this experiment
is a crude analysis of how
well this protein functions
to reduce off-targets.
And these are three guides with
three on/off-target pairs that
have previously been reported
in the literature and out
of the GUIDE-seq work, or the
other high-fidelity protein
papers.
And what we're showing
is that this EMX1 locus,
you see that all four proteins--
wild type, the two literature
proteins, and our protein--
all function pretty well
at the on-target site.
For the problematic
known off-target site,
you can see even wild
type by RNP delivery
does not produce a lot
of off-target editing.
With this assay, we see no
detectable off-target editing
with all three
high-fidelity proteins.
And then interestingly,
if we move over
to this HEK site for
guide, we found, in-house,
that the wild type
protein produces
a lot of off-target editing.
And this is actually a
challenging off-target site
to beat.
It's interesting that we show
that, again, the literature
proteins don't do as well
at this site for on-target
with RNP delivery, whereas
our protein does quite well.
And then all three,
with this assay,
show no detectable
off-target editing.
And it's basically the same
story with this VEGFA3 guide.
Ours looks virtually
indistinguishable
from wild type.
The other high-fidelity mutants
that were developed by plasmids
don't work as well with RNP,
and then all three proteins
reduce off-target
editing below the limit
of detection of this assay.
So this is a very
blunt instrument
for looking at
off-target editing.
Ideally, you would want to do
it in a unbiased, genome-wide
fashion.
So we're going to go back to
this cell-free Cas9 cleavage
system that we've made in-house.
And again, this should
be an overestimation
of the amount of off-target
editing with this protein.
So we think that this is really
putting it through the wringer.
You're not going to have
a degradation of the RNP,
because you don't have
proteases and nucleases.
The genomic DNA is going
to be absent factors that
would encumber Cas9's binding.
You don't have chromatin.
You don't have
transcription factors.
So we tested five
unique crRNAs that
target distinct genomic loci.
And I'm going to show you a
summary of the editing data
from these experiments, and then
hone in and show more detail
on one in particular.
So what you're looking
at here are pie charts
where the blue represents
the fractional percentage
of on-target editing
at these guides.
So with the wild
type Cas9 protein,
you can see we get about
15% of the total reads
are on-target, whereas you
get about 85% here in orange
are the total sum of
off-target editing
events with this particular
androgen receptor guide.
And this is actually
one of the first ones
we looked at in this fashion.
And with relationship to
these other experiments,
it was almost shocking to
see that it only brought
on-target editing up to 52%.
But I'll show you
more detail about why
this is in just a moment.
But looking on some of
these other guides--
the EMX1 guide only edits
on-target 15% of the time.
Our HiFi protein brings
that up to almost 100%.
The beta-catenin
guide we're using here
edits 19% of the
time, on-target.
And ours brings it up to 90%.
This GRHPR guide is actually
pretty good from the start.
It edits on-target
72% of the time.
Our protein brings
it up to 100%.
And finally, this MET guide is
around 29% editing on-target,
and our protein
brings it up to 90%.
What I like about this series
of experiments is, I think it--
if we're anything
at IDT, we like
to be honest arbiters
of the facts.
We want to show things
for what they really are.
And in this experiment,
you see some examples
where you've got a
really poor guide--
and I'll give you more
detail on the specific one
right away-- where our
protein does an admirable job,
but it's not perfect.
And we've also got some guides,
like this GRHPR guide, that
are great from the start,
but our protein just
gives it a little boost.
So let's look a little more
closely at this androgen
receptor guide.
And this is the
same sort of plot
I showed you before, where we've
put the total cell-free editing
sites as a function
of rank order.
And I'm showing you data from
the wild type protein in blue,
and our high-fidelity
Cas9 in orange.
Just to point out,
again, I don't
think that there's anything
from this particular target site
sequence that screams that
this should be a bad guide.
And what we find is that
if we got the asterisk
on the on-target
site, there's not
much difference between the
two proteins for on-target.
But you can see all these
off-target sites in blue,
and the vast majority
of which are eliminated
by using our HiFi Cas9 protein.
So even in one of the
worst case scenarios
where you've got a
really, really bad guide--
most of which, these sites
probably don't actually
happen in the cell--
our protein does quite well.
And we thought that
this was really
putting the protein
through the wringer,
but ultimately, we should
look at this in cells.
So the best way to
do that, so far,
is with the GUIDE-seq approach.
And what we found with GUIDE-seq
is that it does a fantastic job
at identifying what the
off-target sites are
for a given guide.
But where we struggled
to use it in-house
is by giving quantitation
to these numbers.
So we don't find that
these numbers are
very quantitative when we
try to assess them with PCR
amplification-based techniques.
So what we've gone on to do is
to use GUIDE-seq to identify
what these sites are, and then
we use a proprietary multiplex
PCR coupled to NGS
sequencing approach
to actually quantitate
the frequency of editing
at each one of these
different sites.
And so far, we've
done this in-house
with the EMX1 guide that's
been done in the literature.
And I'll show that data today.
And then eventually, we're going
to publish this information
and we'll have a variety
of different guides tested
with this
comprehensive analysis.
And what's so interesting
about this is--
so remember, these
experiments from Keith Joung's
GUIDE-seq work were done
with plasmid delivery
of the guide and Cas9.
And when we did this in-house,
we did it with RNP delivery,
where we know this eliminates
a lot of off-target editing.
And what we found
is that we were only
able to detect editing
at the on-target site
and these top two
off-target sites
when we use RNP delivery
and the GUIDE-seq method.
And this is the data from
that set of experiments.
And we did this with either
the wild type Alt-R S.p.
Cas9 protein or the
new HiFi protein.
And just to point out, this
is a log scale graph here.
And what we see is
that if you take--
with RNP delivery, using this
very quantitative assay--
and look at on-target in orange,
number one off-target in this
light blue, and then the
second-most-frequently-edited
off-target site in gray, you
can see that simply diluting
the RNP complex here gives you
quite a reduction in off-target
editing, and not a huge
effect on on-target editing.
This is basically
what I showed before.
But now if you switch to
our HiFi Cas9 protein,
you only see a
whisper of editing
at this
most-frequently-edited site,
and it's vastly reduced at
this high concentration.
And simply diluting the
complex a little more,
you don't suffer for
on-target editing,
but you eliminate detectable
off-target editing.
So this is very,
very exciting to us.
We are very happy
with these results.
And we actually had been sending
this protein to beta testers,
so we have data--
not just in our hands.
And some of the early
applications of CRISPR/Cas9
are going to be these ex-vivo
genome editing therapies.
And in particular, our close
collaborators Danny Dever
and Matt Porteus at
Stanford are looking
at editing of the
beta-globin 1 gene, which
is associated with sickle cell
disease and beta thalassemia.
And what they have
here in this figure is,
in the upper left-hand
corner-- shows you
the target site they are trying
to edit in this HBB gene.
And what they found
is that there's
a lot of off-target
editing which
was found by GUIDE-seq
at this particular site,
and it is identical
to the on-target site
save for three changes
in the 5 prime-most end
of this target site sequence.
And they find that even with
RNP delivery into primary cells,
there's quite a bit
of off-target editing
at this site.
So looking at the
quantitation by NGS
in three replicates with
the wild type Cas9 protein,
you see greater than
80%-- almost 90%--
editing at the intended
on-target site.
But for this known
off-target site,
you can see the values range
from 30% to 50%, so quite a bit
of concerning
editing at that site.
And as you can
imagine, this would
be a challenging
off-target site to beat,
since the changes
are so far away
from the PAM or the SEED
region of the guide.
So we sent them our HiFi Cas9
protein, and what they show
is that they largely
maintain on-target potency
with our protein, and they
reduce off-target editing
significantly.
And over here's a
quantitation of that data.
You can see, with
our HiFi protein,
they maintain 75%
to 80% editing,
and they've reduced 30% to
50% down to 1% to 3% editing.
So they see a really
strong reduction
in off-target editing.
Many of these other sites that
were either found in GUIDE-seq
or found via prediction are
sort of functional zeros.
But there are a
couple examples here
of sites that are edited
0.6% to 0.2% of the time,
that our protein also
reduces to a functional zero.
And what I think is
also very exciting
about this is not only do
they look at indel formation,
but they also looked at HDR--
or homology-directed repair--
in primary cells at this
locus, and they see
that they maintain
approximately the
same amount of HDR
using our HiFi Cas9 protein.
And then they've also
gone on-- so this
is the indel formation through
these other clinically-relevant
guides that I've already shown.
They've also gone on
to do HDR experiments
at all these different
clinically-relevant loci,
as well.
And they show basically
the same story
that we've been
saying all along--
is that our protein maintains
a lot of its on-target potency,
even with HDR, at these
different clinical sites.
And the other
high-fidelity proteins,
which were made for
plasmid delivery,
seem to show a marked
reduction in HDR.
And we don't have any
off-target data yet
to look at our HiFi
protein in mice.
That's something we're
working on pretty hard.
But we have taken a look at
how well it functions on-target
at several different sites
within several different
genomic loci, and we're very
pleased to find that it seems
to work on-target very well.
We don't expect any
surprises for off-target,
since we've already looked at it
in a totally cell-free context.
And with that, I want to make
a few concluding statements,
and then I'll end
on one experiment
that I think pulls
everything together.
So off-target effects are a very
real concern when using CRISPR.
They're potentially
a hidden disaster.
Simply switching to RNP
delivery can get you
a good bit of the way there to
reducing off-target effects.
A lot of the mutations in Cas9
that have been made to reduce
off-target editing--
depending on the
delivery mechanism,
these can also hurt
on-target cleavage.
We've used a bacterial
selection scheme
to find a protein that maintains
the best on-target editing
while reducing off-target risk.
And I think, just
to be practical--
I mean, there's
no way that we're
going to ensure 100% on-target
and completely eliminate
off-targets.
But I think you can get part
of the way there by using RNP,
carefully selecting
a target site--
and hopefully we can
develop better algorithms
in the future--
and finally, by using
high-specificity enzymes
like our Alt-R HiFi.
And I think that this experiment
pulls everything together.
And this is, again, an
editing experiment with EMX1.
And what you're looking here is,
again, at a log scale graph--
looking at the on-target
site, the top off-target site,
as well as the
second-most-frequently-edited
off-target site.
And you're looking,
in dark blue,
at the wild type Cas9
produced in a stable cell
line, where you expect
off-target editing to be high.
You're looking at wild type
Cas9 delivered as a 2 micromolar
RNP, and then our HiFi protein.
And you can see all
three of these proteins
and delivery mechanisms give
you good on-target editing.
You can achieve that any way.
But if you look at
off-target editing,
you get the highest amount with
Cas9 produced in a stable cell
line.
Simply switching to RNP
delivery for either site
gets you a good deal of the way
towards reducing off-targets.
And then finally, switching
to our HiFi protein
and diluting and doing
a careful dose response
gets you the rest
of the way there.
So we're very, very
excited about this product.
It's been a long
time coming, and I'm
glad we're finally able to
release it onto the market.
And I'd like to thank
everybody for listening in,
and I'd be happy to
take some questions.
I guess it's not
surprising that you're
excited about that, since
you've done so much work on it.
[LAUGHTER]
There's that.
Hey, so, we have plenty of
time for questions here.
And if you have a question
for Dr. Vakulskas,
you can ask them right now.
You can type them into
the Questions box,
and we will read through as
many of them as we possibly can.
I also wanted to
draw your attention
to the chat box really quick.
We've posted the slide deck
for today's presentation,
and you can grab that right now.
If you don't want to
look at it right now,
we will also be sending
you a link to it by email.
So you don't really need
to worry about that.
Just-- it's available
to you if you want it.
OK, so here's a
question-- and this
is a great question, just
in the context of all
of this-- talking
about design algorithms
and the need for a HiFi Cas9.
It used to be that there
was a very clear idea
of the importance of the
position of those variation
bases.
If there's a variation that's
far away from the PAM site,
it doesn't have as much of
an effect as if it's close.
But is that difference
also hard to predict now?
So I mean, the
truth of the matter
is I just don't think
we have all the data we
need to make those conclusions.
But what I will say
is I suspect it's
a combination of multiple
things happening at one time.
I think the traditional
rules apply,
where the closer a change is
to the intended target site,
the closer it is to the
PAM or the seed region,
the less likely it is to
be an off-target site.
But I think you probably
have other factors like--
and this is just speculation--
some basic cleavage preferences
for the Cas9 enzyme.
So these are things that happen
when Cas9 already gets there.
Maybe it has a preference
for a specific nucleotide
at a specific position.
And under the right
circumstances,
that can trump simple
binding interaction kinetics.
I just think there's a lot of
things probably happening--
to borrow the phrase--
in a mosaic, and
it's really hard
to decipher what those
rules are, and so forth.
Sure.
OK, so I'm not sure how much
of this you can talk about.
But I'll ask the
question anyways,
and maybe you can give a
little bit of detail here.
How many mutations does
the HiFi Cas9 have?
Yeah, so that is something I am
not able to talk about today.
But what I can say is
that we are, at heart,
an academic organization.
That's how we were founded.
And we do intend to publish
this as quickly as possible.
And obviously, we're
going to identify
what the position or
positions are at that time.
OK.
Yeah, no, that's a--
Sorry.
--good answer.
Actually, it's nice
that we are planning
a publication for that.
Yeah, absolutely.
This is a question that we
do get from time to time.
I'm not sure--
I haven't heard a
recent answer for this,
so it's good to be
asking it again.
Do we have any
recommendations for people
for applying RNP delivery
in plant genome editing?
Yeah, so that is something
that our protocols are not
quite there yet, on.
But it is actually-- so that did
come up at a recent conference,
and it's something that we're
working very hard to address.
And I think that what's
interesting is a lot of people
that are working in protoplasts,
they're finding that
plasmid-based delivery of Cas9--
the protein isn't
made fast enough
for how quickly you have to
manipulate the protoplast.
You need Cas9 expression.
You need the editing
to happen quickly.
So people tend to,
apparently, favor RNP delivery
in these particular protoplasts.
So that's become something
that's of high importance
to us, and I think we should be
able to address it, hopefully,
in the near future.
OK.
This is interesting.
Do we have anything about Cas13?
Are you familiar with that?
Yeah, so this is the-- if I'm
not mistaken-- the RNA editing
technology that's been applied
to a diagnostic setting.
We don't have a product
for that at the moment,
but we always have
our ears open to
new and exciting technologies.
And obviously, there have
been a couple great papers
about this technology,
and it's something
that we're looking
at very carefully.
I apparently need to
go do some reading.
[LAUGHTER]
Let's see, here.
There's a few similar questions.
People really want
to know about--
they want to know the details
of the enzyme itself--
sequence and
changes and whatnot.
So yeah, look for that paper
in the future, coming from--
Dr. Chris Vakulskas,
apparently, will
be one of the authors on there.
Hope so.
[INTERPOSING VOICES]
[LAUGHTER]
I would assume.
I would assume.
But really, we want
to publish this
as quickly as humanly possible.
So that information is
going to be out there.
OK, so this must be for the
staph aureus, I'm guessing.
Do we have HiFi staph aureus--
SA-- Cas9 doing a similar job?
You know, we
certainly don't even
have a commercially available
wild type staph aureus.
And I think, if memory serves,
that has a more restricted PAM
site, so that's not
something that we've
done a lot of testing with.
But I can't say for certain, but
that might be the sort of thing
where we'd be open
to looking into that.
I expect the amino acid
positions are likely conserved,
so.
Yeah, we may be open to that.
But right now, we don't even
have a wild type SA Cas9.
OK.
Have we contemplated
working with the HiFi Cas9
to create nickase variants?
Yeah, that's an
interesting idea.
We've also thought
about the dead Cas9
and introducing a HiFi
in that context, as well.
But our thinking on that so far
has been, with the nickases--
in doing HDR with
these dual guides,
we didn't see the need to
increase the fidelity--
since you're only
nicking and the only way
that you're going to get an
actual double-stranded break
is if you have two
entirely separate guides
in close proximity.
So I guess what I'm saying
is the nickase-based HDR,
or cleavage approach, is
inherently high-fidelity.
And we haven't, so far, seen the
need to increase its fidelity.
But again, we're always
open to this stuff, so.
Right.
And did you want to say a
little bit about the nickases
that we also have?
Yeah, so at the same time we
released the high-fidelity Cas9
enzyme, we also began selling
the D10A and H840a nickases.
We actually have
some good protocols
for those available
on the web, so feel
free to visit the website
and look at the data.
And if anybody is looking
for them, we have them.
OK.
This is an interesting one.
So have we tried plasmid
delivery of the HiFi Cas9?
And I think you
touched on that a bit.
And then there's a
second part, which is--
how does it compare to eSpCas9?
Yeah, so we have tried
plasmid delivery.
And in fact, when
you're doing screening
and that sort of
thing, it's just easier
to do all this stuff on plasmid.
But the caveat there is--
when we were doing
those experiments,
we supplied the guide RNAs
with our two-part Alt-R system.
So we didn't supply
those on a plasmid.
So with that
approach, our protein
actually worked pretty well,
even with plasmid delivery.
But honestly, we didn't do a
real careful comparison of it
to the other
high-fidelity mutants,
because our main interest
was looking at it
in the context of RNP delivery.
But I can say it's not like ours
just doesn't work with plasmid.
I suspect where it
would not do as well,
relative to the
other high-fidelity
proteins, is if
you were delivering
both components by plasmid.
And in that case, I think the
other high-fidelity proteins
are probably a
better option anyway,
because they were specifically
evolved for plasmid, so.
So I've been doing these
webinars for a while,
and I'm going to tell
you that I have thought
a lot more about plant
stuff through this process
than I would have otherwise.
So this is how I get
educated about plant stuff.
Somebody is asking-- and this
is probably going to be out
of your area of expertise, but--
is there any reason
why off-target activity
would be less important for
plants, that you can think of?
I just-- I don't know
enough about how easy
it is to breathe out
unwanted off-target edits.
And I think, going
back to the idea
that delivery
mechanism matters, it's
possible that either the
Cas9 protein or the guides
are less stable in
particular plant species.
Maybe they're more rapidly
diluted out by growth--
although that's kind
of hard to imagine.
I'm sure there are some cell
lines for some organisms where
the RNP doesn't hang
around very long at all.
Maybe it's very,
very short-lived.
And in that context,
you would imagine
that you would get the lowest
amount of off-target editing.
But unfortunately, I just
don't know enough about plants
to intelligently
answer that question.
Right, right.
And like I said, I
mean, I've certainly
become more thoughtful
about plant questions,
because we do have so
much interest in that.
Obviously, agricultural research
is a really important area,
but that's not where
I'm trained, so.
Absolutely.
I mean, this is a
question we can possibly
ask to some other people.
We happen to have a
product manager who
has a PhD in plant
biology, so that
might be a question for him.
All right, another question
here that I'm not quite sure of.
It says-- just to clarify,
targeting a single base pair
mutation for repair
in a cell line
is not exactly
feasible at the moment?
And that's a statement with
a question mark at the end.
Could you repeat that?
I'm not sure I fully understand.
So I'm thinking that what
this person is asking is--
so it says, targeting
a single base pair
mutation for repair
within a cell line
is not exactly
feasible at the moment?
And I'm guessing that
they're saying that--
sure, you can correct that.
But you can't really do it
precisely, at the moment,
with the current technology.
Well, I mean, all I
can say about that
is it would be difficult to
select for such an event.
I mean, ultimately,
what you would
have to do is get an HDR--
I mean, I can think of
two strategies to do it.
One is HDR.
And since there would not be
any selection for a single base
change--
because obviously,
you can't incorporate
an antibiotic resistance
marker or anything--
you'd have to have a pretty
high-frequency HDR event,
and then be able to isolate
the clone by limiting dilution.
But the other way is-- there
are also these so-called base
editing enzymes that have
come on the forefront,
where you're taking a dCas9--
so a dead Cas9--
coupled to a cytosine deaminase.
And with those particular
recombinant forms of Cas9,
you can actually introduce
a specific point mutation
in the absence of HDR.
So I mean, I think
it is possible.
I just don't think
it's easy yet.
Right.
Yeah, that's interesting.
OK, so do we have any
recommendation for finding
specific guide RNAs in plants
without the total genome being
sequenced?
Oh, boy.
You know, I don't know what the
best answer to that is other
than, empirically, trying to
empirically determine what
the total number of
off-target sites is--
and the existing
technology, obviously,
the majority of
what's done is done
in immortalized cell lines.
So I don't know how
easy it is to do
a GUIDE-seq in plant cells.
I do think the available
algorithms are going
to help you identify the most
obvious off-target sites,
and I think that has to be
a component of every one
of these experiments.
And I think eventually, the
algorithms will get better.
And I mean, I guess my
only recommendation would
be to try to do it empirically.
And something like
a GUIDE-seq would
allow you to do that without
sequencing the entire genome.
Hey, here's a completely
interesting question,
and it's not exactly science.
I haven't thought about this.
Do you know, does somebody need
licenses to use our enzyme?
I think when you purchase it,
you're acquiring-- and I mean,
don't quote me on this-- but
I believe you're acquiring
an individual use license.
But I mean, I guess it
depends on who it is, right?
I mean--
Exactly, so--
If you're talking about
on a commercial scale,
that's probably a
different story.
Here's the thing-- yeah.
If you have this question--
I mean it is, I think,
going to depend somewhat on,
yeah, what you
plan to do with it.
So if it's another company
that plans to use this,
it's possible.
Yeah, if you're trying to use
it outside of the research
use only that we
offer it for, then
you're definitely going to
want to talk to somebody here.
You can find the
right person at IDT
to talk to by going
to our website--
idtdna.com/contactus-- and
somebody can help you talk
to somebody specifically about
a legal concern like that.
But for the most part,
for just research use--
yeah, if you bought
the enzyme from us,
you can definitely use
it for basic research.
We'll definitely follow up
with this particular question.
I haven't seen that
before, but it's obviously
a concern to people.
Yeah, absolutely.
OK, let's see here.
This next question is-- have
you done additional rounds
of directed evolution for
protein engineering using
your--
using your surprising
finding of androgen receptor
off-target activity?
So maybe, have we used
that guide as something
to pan the directed
evolution against?
Oh, sure.
So they're asking-- and that
is the rest of the question
that they're
asking, is-- can you
make an even more
high-fidelity Cas9?
Yeah, I mean, I
suppose it's possible.
The trouble we've run into is
the more we mess with things,
the more we reduce
on-target activity.
And you very quickly lose
significant on-target activity.
And I think we put a lot
of effort into making more
specific versions that
ultimately, we ruled out,
because they just lost too
much on-target activity, and--
You're looking for
the best balance.
[INTERPOSING VOICES]
Yeah, the best balance.
And I think that's
what we found here,
and it's not something that
we're ruling off the table.
And in fact, that's a good idea.
That androgen receptor guide
would be a good one to look at.
OK, this is a good
question, too.
What is the impact of the
cell doubling time for the RNP
efficiency, and is it still
working with cells that
have a week of doubling time?
Man, that's just something I
don't think we've looked at.
That's a good question.
We haven't looked
at that carefully.
I can say that RNP delivery,
the cells are very--
it's more finicky,
especially with lipofection.
So you need to take cells that
are not fully confluent yet,
and that sort of thing.
But we really haven't
done a careful analysis
of cell doubling time
and editing efficiency.
We generally look at the
results of our editing,
though, at 48 hours, right?
Mm-hm.
So I mean, maybe there's
a partial answer in there.
For us, we get our editing
results within 48 hours.
Maybe that's a helpful
way to gauge that.
Yeah.
Yeah, and you know, I think
we need to go out to 48 hours
to see the highest
levels of editing.
But also, I wonder
if that's going
to depend a lot on the cell
line and species as well, so.
OK.
OK, here's a question for you.
And it says, what is the
recommended guide length
when using the HiFi Cas9?
So for the Alt-R system, we're
recommending 20 and no shorter.
And so that's the guide length.
But also, I mean, we
really do recommend
using the two-part guide
RNAs that we offer.
Just because, I
mean, they've also
been optimized for other things,
They're length-optimized.
They have the
modifications and whatnot
to prevent that immune
response and such.
That's why we really
do recommend those.
Right.
So yeah, so it's 20
nucleotides of guide sequence
give you the absolute
best results.
OK.
So all right, we've reached
the top of the hour.
There are still a few questions
that have not been answered,
and unfortunately, we
can't fit them all in.
But we definitely will follow
up with everybody by email.
If you feel like you would
like to talk to someone,
you can go to that
idtdna.com/contactus--
all one word, no spaces, no
other characters in there.
And just yeah, reach out.
Otherwise, yeah,
we'll be emailing you,
and you should get an
answer to your question.
You'll also be getting
a follow-up email
with links to the presentation.
And the slide deck
is already available,
but you'll get links to
the slide deck as well.
And really, thanks everybody.
Really great questions
for this Q&A session.
And Chris, it was an excellent
presentation, so thanks to you,
as well.
Yeah, thanks everybody.
