Hello and welcome to this
Integrated DNA Technologies
webinar, "Increasing
Genome Editing Efficiency
with Optimized
CRISPR-Cas Enzymes."
My name is Sean
McCall, and I will
be serving as moderator
for today's presentation.
Today's presentation
will be given
by Dr. Cristopher Vakulskas.
Chris is a senior staff
scientist in the Molecular
Genetic Research Group at IDT.
At IDT, Chris has managed
contract research projects,
led process development for
CRISPR protein purification,
and developed novel CRISPR
proteins, including Alt-R Sp
HiFi Cas9 Nuclease 3NLS.
Chris's presentation should
last about 30 minutes,
and following the
presentation he
will answer as many questions
as possible from attendees.
The question and
answer session will
be conducted by Ashley Jacoby,
staff scientist at IDT.
As attendees, you
have been muted.
But we encourage you to ask
questions or make comments
at any time during or
after the presentation
by typing into the questions
box located in the GoTo Webinar
control panel.
Also, in case you need
to leave early or want
to revisit this webinar, we
are recording the presentation
and will make the
link to the recording
available on our website a few
days after the presentation.
We will also post the
recorded presentation
on our YouTube
and Venue channels
and post the slides on
our slide share site.
You'll receive links to
these in a follow up email.
So now let me hand it over to
Chris for his presentation.
All right.
Thanks Sean.
Good afternoon everybody.
I'm going to talk to you this
afternoon about some work we've
done to increase the activity of
our entire suite of CRISPR Cas
enzymes.
And I'll start by
just giving you
a basic overview
of genome editing
and some of the
products we offer.
And then I will
move into the idea
that the delivery mechanism
of the CRISPR enzymes
affects toxicity and
off-target effects.
I'll spend an awful lot of time
talking about the Alt-R HiFi
protein and how it can be used
to reduce off-target effects.
And then after each
section I'll try
to go into some
detail about how we've
improved each one
of these enzymes
and show some real data.
Finally, I'll talk
about our Cas12a,
otherwise known as Cpf1
protein, the basic workings
of that system, and then end on
the various improvements we've
made to that system and,
again, show some data.
So just to get started,
essentially what
we're looking at are
two different RNA-guided
endonucleases that are used to
edit genomes in living cells.
And you're trying to introduce
a double-stranded break, as seen
at the top here, and generally
do one of two things,
either facilitate
homology directed repair
with some sort of DNA
donor, totally replace
the edited locus, or,
on the right-hand side
here, allow the non-homologous
end-joining pathway
to form an indel, usually
used for gene disruption
and so forth.
And arguably you could
add a third branch on here
for the newly developed
base editors, for both Cas9
and Cpf1.
And I'll start by just going
over the basics of Cas9, again
an RNA-guided endonuclease.
Here you're trying
a 23-base sequence,
20 nucleotides of which
are encoded on the RNA.
The PAM site for Cas9 is NGG,
so anywhere on the genome
where there's a G
dinucleotide repeat,
theoretically you could
have a targetable Cas9 site.
Cas9 exists in bacteria as part
of a primitive immune system
where you have a targeting
CRISPR RNA or crRNA
and a universal tracrRNA that
hybridized together to form
the functional guide RNA.
Those have also been
appended together covalently
to form a single guide or sgRNA,
and this cartoon at the bottom
depicts a functional
RNP, or ribonucleoprotein
complex, where you
can see that Cas9
in that sort of light
blue color with the tracr
and cr in a
[INAUDIBLE] together,
bound to the target site.
And as I mentioned,
you can use this
as either part of the
native two-part system
or as the artificial
single guide RNA.
At IDT, we actually favor
the two-part system,
for a variety of reasons.
Here you've got two
very short RNAs,
which are not ideal, to express
as part of a plasmid delivery
vector.
They're really just too short
to effectively terminate.
And for the same
reason, they're not
ideal for in vitro
transcription as well.
However, we are an
Oligo synthesis company,
so we can make them very
easily and inexpensively
by chemical synthesis.
So for the CRISPR
RNA or crRNA, you're
talking about a 36
mer that's comprised
of 20 bases of a
targeting sequence with 16
bases of constant [INAUDIBLE]
to anneal to the tracr.
The tracr RNA is a bit longer.
It's a 67 mer.
But it's still very efficient
for chemical synthesis.
Because we're making them
chemically one base at a time,
we can actually incorporate
modified bases quite easily.
So it's easy and cost
effective to modify.
That allows us to
introduce modifications
that help escape nuclease
degradation as well
as the immune response.
And at the bottom
of this section,
you just kind of see the crRNA
up top annealed to the tracr.
And alternatively, with
the single-guide RNA
on the right-hand
side, these are
ideal for DNA
expression cassettes,
because they're a bit longer.
This is approximately a
100 mer, so it's easier
to terminate these as part of
plasmid-based delivery systems.
For the same reason,
they're ideal for in vitro
transcription.
You can transcribe
them in your lab
and introduce these into cells.
I would say they are relatively
low cost to produce that way.
Chemically, because
of their length,
they're relatively
inefficient to make.
Well, we can make them.
Hence the higher cost.
They are costly to modify,
again, because of the length.
And if you're going
to produce them
with in vitro
transcription, it's
very, very difficult or
impossible to modify,
depending on where you're trying
to introduce the modification.
And we've also found
that sgRNAs produced
through in vitro
transcription can also
cause an awful lot
of toxicity, which
I'll show in the next slide.
But I point out before I move on
that the structure of these two
things are very,
very similar, and all
that's missing with the two-part
system are the covalent links.
So just taking a really broad
overview of what we found
happens when using in vitro
transcription produced sgRNA,
we're just simply looking at
the cells under a microscope
and looking at
morphology in abundance.
And if we look at the
left-hand side here,
this is simply a microscope
view of confluent HEK-293 cells.
They look very healthy, and
these cells are constitutively
producing Cas9.
In the middle, we've
transfected 30 nanomolar
of an IVT-produced sgRNA.
And you can immediately see the
lack of cell abundance and lack
of proliferation,
indicating there's
some toxicity with
this experiment.
Now if you look at
the right-side panel,
here we've taken the
same targeted guide,
but instead of an
sgRNA, we're using
a chemically synthesized
two-part system.
And you can see
these are largely
indistinguishable
from the cells only.
And I point out that despite
the differences in toxicity,
both of these experiments
produced equivalent levels
of gene editing.
So they both work,
it's just that we
see evidence of toxicity
for the IVT-produced sgRNA.
And looking a little more
closely at the transcriptome,
on the left-hand side
here what we're looking at
is a qPCR assay for a human
normalizer gene SFRS9.
This is something
where-- a control, where
we don't expect the levels
to vary no matter what we do.
And you can see all of the
data points are on top of one
another-- the IVTs, the
two-part, and the cells
only, exactly as
we would predict.
But if we look on
the right-hand side,
we're looking at a
marker for stimulation
of the immune system,
IFIT1, and you
can see that the two-part
system and cells only
are indistinguishable,
suggesting
there's no induction
of the immune system.
However, there is a massive CQ
shift to the left for the IVTs,
indicating that the immune
system is in fact stimulated
when you use these RNAs.
And IFIT1 is just one
example of several markers
for the immune system
that we've look
at that show high induction
of IVT-produced sgRNAs.
And we just don't see this
with the two-part system.
Nevertheless, there are
a lot of different ways
to implement CRISPR Cas9 genome
editing in your laboratories.
On the left-hand side,
looking at the Cas9 proteins,
starting from top to
bottom you can purify Cas9
in your own lab.
You can purchase
it commercially.
That can be delivered directly.
You can either make or acquire
a Cas9 stable cell line
where the Cas9
protein is expressed
at a constitutive
level all the time.
You can purchase or synthesize
in your laboratory a Cas9 mRNA.
This can be transfected directly
with the right protocols.
And probably the
most popular is using
a either plasmid or viral
vector-based DNA delivery
system at the bottom.
And it's essentially the
same story for the RNAs.
If you look at the right, in
green, from top to bottom,
again at IDT we favor
the two-part system
made by chemical synthesis.
You can also produce these
by [INAUDIBLE] transcription
to make an sgRNA.
That does work.
You can supply a DNA
fragment, be it a PCR product
or a gBlocks gene fragment
that encodes the RNAs that
can be transfected and produce
a successful genome editing
experiment.
And lastly, much
like Cas9 protein,
you can supply the RNAs off
a plasmid or viral vector.
These can be mixed and matched
however you want really,
and you'll get genome editing
to work on some level.
At IDT, however, we tend
to favor ribonucleoprotein
delivery.
And that is taking purified
Cas9 and chemically synthesized
two-part, putting them
together in a test tube
to form the RNP complex and
then delivering that directly
into cells.
And part of the reason
for that is simply
just because of how easy it is.
And it's a three-step process
for Cas9, where at the top
you take the universal tracrRNA
and the specific CRISPR
or crRNA, add them together
in a test tube, heat
and slow cool to form
the guide RNA complex.
That is added directly
to purified Cas9 protein
at a one-to-one ratio.
The RNP is formed after about
10 minutes of room temperature.
And then that can be
delivered directly
into cells by whatever your
favorite delivery mechanism is.
And we have protocols online
for lipofection-based delivery.
We have protocols
for electroporation
with either the
Amaxa Nucleofector
or the Neon system.
There are also some user
protocols for microinjection
and other more niche systems.
Again, all of these can be
accessed from the IDT website.
So it's very easy to use.
But probably a more
important reason to use RNP
is because of the propensity
for Cas9 to cleave off-target.
And I think that there is
an awful lot of confusion
in the field as to how
much a person should
worry about off-target.
So a lot of that,
I think, is going
to depend on how you
set the system up,
what guides you've chosen,
and then also just, you know,
what type of work you're doing
and how much off-target editing
events are going to matter.
And I think, whatever
you're doing,
there are some very
simple ways to mitigate
the risk of off-target events,
starting with using RNP.
And what you're
seeing on the screen
is an example from the
GUIDE-Seq approach.
This is an approach
that's widely
been employed to determine
the total repertoire
of edited sites for any
given CRISPR experiment.
In this case, the authors are
using an EMX1 targeting guide.
This is a very popular
site in the literature.
And they've got all these
different sites listed.
So this is an
NGS-coupled approach,
so the number of
GUIDE-Seq reads or NGS
reads that correspond
to the number of indels.
So at the top with
this filled square
is the number of
reads associated
with the on-target site.
And thankfully it's edited
the most frequently.
And then everything
at the bottom,
moving down from top to bottom,
are all the off-target sites
and the frequencies with
which they are edited.
What I think is kind
of scary about this is,
if you look at the top two
sites beneath the on-target,
these are edited in
near equal frequency
to that of the on-target site.
And for certain applications,
that can be kind of scary.
And I think one of the
things to keep in mind
with this experiment is
that the authors were
using plasmid-based delivery of
both Cas9 as well as the RNAs.
And we have found that to
be a condition where you get
the most off-target editing.
And I can give you a lot
of our own internal data,
but I think it lends a lot
of trust if I can show you
data from other
companies and then
show that it falls in line
with the data we've produced.
So our friends at Thermo have
shown this nice experiment
where they vary the delivery
mechanism of Cas9 protein.
And they essentially show that
the longer it sticks around
in the cell, the more
off-target editing you get.
So Cas9 protein by
Western Blot on the left,
and the ratio of off- to
on-target on the right.
These are two known
off-target sites
for the guide that was used.
So going from top to
bottom, with a DNA-
or plasmid-based system,
they find that Cas9--
it takes a while for it to reach
a steady state level of cells,
about a full day,
and then it doesn't
seem to reduce it all all
the way out to 72 hours.
And not surprisingly,
in black you
can see that the DNA-based
delivery mechanism
is synonymous with the most
off-target editing seen
for both of these sites.
If you look at mRNA,
that seems to show
that Cas9 protein appears a
lot sooner, about four hours,
and the levels do not dissipate
that much, even all the way
out to 72 hours,
although arguably there
is maybe a two-fold decrease.
And that's sort of
an in-between level
of off-target editing between
DNA and protein here in blue.
Protein, however-- so if you're
transfecting Cas9 proteins
directly, all of it seems
to show up immediately
after four hours.
And then it just dilutes
or degrades by growth
beyond that point.
And by 72 hours it's
virtually non-existent.
And not surprisingly, in
this red or burgundy color,
RNP-based delivery is consistent
with the lowest amount
of off-target editing seen.
So, you know, we talk
about it in the lab
as sort of a fast on,
fast off approach,
where the longer Cas9 is
around to cause mischief,
the more off-target
events you get.
So, yeah, that's the
same sort of experiment
that we show in house.
And what we've done
here is to compare
Cas9 that is expressed in a
constitutive stable cell line.
HEK-293 cells have
always make it
to that of
ribonucleoprotein delivery.
And the readout for
this, in dark blue,
this is the on-target
site for this guide.
So we're getting
very good editing.
And then these in
light blue and gray
are two known off-target
sites for this guide.
And you can immediately see
that on-target editing is
very similar between
the highest doses of RNP
and for the stable
cell line, yet there
is a lot more off-target editing
seen for the stable cell line.
And you can actually dilute
out the RNP quite a bit
here without sacrificing much
on-target editing efficiency.
And yet the off-target
events go down even further.
It is only after this
half micromolar dose
before you start to see a
loss in on-target editing
efficiency.
So our finding is that simply
switching your delivery
mechanism to RNP can
mitigate a lot of the risk
from off-target editing.
But we've also
found that there are
a lot of cases where, despite
the fact that you switched
to RNP delivery, there are
certain persistent off-target
sites that show up no
matter what you do.
And also if you're
somebody that's
working in the
therapeutic realm,
even minute quantities
of off-target editing
could be a real problem.
So what do you do?
And one of the approaches
to deal with that
has been to try to predict
off-target editing based
on the guide sequence
you've chosen.
And these are two examples of
the more popular prediction
logarithms, the MIT algorithm
and the Heidelberg CCTop.
And what we've found
is that it is just
an extremely difficult
thing to accurately predict
Cas9 cleavage sites.
And for that reason, we do
not think that this approach
is quite ready for prime time.
And here's some
data to demonstrate
what I mean by that.
So for this particular androgen
receptor guide sequence
that I'm showing
at the top here,
there's nothing about this
sequence that seems repetitive
or like it would
be a huge problem.
But nevertheless, if we
look at the total repertoire
of edited sites for a given
genome editing experiment,
we see an awful lot
of off-target cleavage
with this guide.
So on the far left
in red, that's
the frequency of editing
seen with the on-target site,
and everything in blue and
green is an off-target site.
So there's quite a bit of
off-target editing there.
And I've coded in
green the sites
that are predicted by any
SVM prediction algorithm.
And what's scary
is that you find
that there are only six
sites here in total that are
predicted, representing
less than 10%
of the total off-target editing.
And I'd also point out that
the top most frequently
edited off-target
sites are not predicted
by these algorithms at all.
These are probably the ones
you care the most about.
And I don't think
that, you know,
this is to say that
all the algorithms are
bad or poorly constructed.
I think it's more
evidence that it's
an extremely
difficult thing to do,
to predict off-target editing.
And it's just, you know,
we're not there yet.
I think one day we will be.
But in the meantime,
what more can you
do to minimize the risk
of off-target editing?
Well, first of all,
changing delivery mechanisms
is important.
Second, there have
been some other things
that have come up
in the literature
to solve this
problem, one of which
is reducing the length of the
crRNA from 20 mer to 19 or 18.
We've tested this
internally, and we
found that in certain
cases it can indeed help,
but oftentimes you'll find
an unpredictable reduction
in on-target editing
at the same time
as a reduction of
off-target editing.
That's something you don't want.
It's been basically
the same story
for chemical modifications.
There have been some
papers that have
come out saying if you
put specific altered bases
at specific positions
in crRNA you
can minimize off-target editing,
and we find it's a mixed bag.
Finally, one of the
things that has come out
is the notion of
mutant Cas9 proteins
that are so-called
high fidelity Cas9s.
And these are two
of what I think
are approximately five mutants
present in the literature
right now--
the eSpCas9 (1.1) protein
from Feng Zhang's lab
and the SpCas9-HF1
from Keith Joung's lab.
These fantastic
studies done, where
they looked at the
crystal structure of Cas9
and tried to rationally engineer
alanine substitutions that
would reduce the affinity
for off-target sites
and hopefully not for
the on-target site.
And we had set out at IDT to
test these mutants, because we
knew off-target
editing was a problem,
make them in the
context of our Cas9,
and test for off-target cleavage
as well as on-target cleavage
with RNP delivery, with the
intent of finding the best
and hopefully licensing
and commercialize it.
And we started by testing
some of the guides that
are frequently looked
at in the literature--
the EMX1, HEKSite4,
and VEGFA3 guides.
And we looked at both on- and
off-target cleavage for both.
These represent just a single
off-target site for both.
And we found that
for EMX1, all enzymes
were comparable to Wild
Type for on-target.
And all of them reduced
off-target to undetectable
levels, which is very good.
But when we moved on to the
HEKSite4 and VEGFA3 loci,
we started to see real problems
with on-target editing,
despite the fact that
they were both good
at reducing off-target.
So this is quite worrisome.
And we thought,
OK, well maybe we'd
better take a broader
survey of sites
and find out how big
of a problem is this.
And so we looked at
several different sites,
at the [INAUDIBLE]
PDCD1 loci, and we
found that these enzymes really
struggle to edit on target
when using the RNP
delivery platform.
And, you know, I would point out
that these enzymes were evolved
under conditions where they
have plasmid-based expression
of both Cas9 as well
as the guide RNA,
so they were never made
to work for RNP delivery.
So I don't think it's a
failing of these mutants,
I just think we put
them in a situation
where they were not
designed to work that way.
I think, with plasmid delivery
they probably work quite well.
But for RNP we just needed
a different solution.
So these mutants
were selected based
on plasmid results,
where you have continued
and long-lasting
Cas9 synthesis that's
always being re-expressed.
We know that plasmid delivery is
prone to toxicity and problems
with the immune system,
so we don't favor it.
We like RNP.
And we found that
no existing HiFi
mutant works as well as RNP.
And that includes more than
just eSpCas9, SpCas9-HF1.
We've also looked at HiFi
Cas9, we've looked at Evo Cas9,
we've looked at xCas9.
And we always find
the same thing--
a reduction in
off-target editing
at the expense of on-target.
So we developed in-house a
proprietary bacterial system
to find our own HiFi mutations.
And what we did
differently is that we duly
selected for reduction in
off-target editing as well
as the maintenance of
on-target efficiency.
So selecting for both
things at the same time.
And this is a basic
cartoon demonstrating that.
And I won't go into
all the details here,
but essentially what you've
got is a high copy plasmid that
expresses a bacterial toxin.
So if this plasmid survives,
the bacterial cells are killed.
And on this plasmid is an
on-target site for the guide
we're using.
So we're asking
CRISPR Cas9 to cleave
every one of these plasmids so
that the toxin is not produced.
And simultaneously, we
have a second plasmid,
an off-target site
containing plasmid,
where you need this
plasmid present to survive,
since it expresses the
antibiotic marker or resistance
marker, but it also has
this off-target site.
So Cas9 has to
cleave all of these
and avoid cleaving the majority
of these off-target site
plasmids in order to survive.
And it's important to note that
the Wild Type Cas9 does not
pass this screen.
It would not facilitate
any survival.
So we made a random
mutagenesis library of Cas9,
passed it through this screen,
pooled all of the mutants,
changed the guide and target
sites, did the same thing.
And ultimately we
settled on a handful
of mutations, evaluated
them very carefully,
and settled on one very specific
combination of mutations that
resulted in what we're now
calling the Alt-R sP HiFi Cas9
protein.
Here's some data in two
very different systems
to show you the on-target
performance for this HiFi Cas9
protein.
And on top we've got the
two-part RNA system, delivered
by RNP lipofection, testing
12 different guides that
target the HPRT locus.
These are in standard
HEK-293 tissue culture cells.
And we're comparing in dark blue
the Wild Type Cas9s to our HiFi
Cas9 in orange, and then the two
other literature high-fidelity
proteins are in that
light blue and gray color.
And you can see at the
vast majority of sites
the orange HiFi works
nearly as well or as well
as the Wild Type protein,
where the other literature
proteins really struggle.
And if we look at
the bottom here,
this is an experiment
done by our collaborators
at Stanford, the Porteus
Lab, and they're using
chemically modified sgRNAs.
They're delivering these RNPs
into cells via electroporation.
And they're looking
at human primary cells
and targeting four different
clinically relevant loci.
And then, whereas
we were detecting
with T71 cleavage, at this
[INAUDIBLE] right here
we're looking at NGS.
So very, very different
system demonstrating
essentially the same result.
The Wild Type and IDT
HiFi worked very, very
similar at most of these sites
where the other
literature HiFis fail.
So these things are-- this HiFi
protein works great on-target.
But it's probably good to
show some off-target data.
So these are three sites
tested in literature.
And we first looked at
single off-target sites
to see what happened.
And EMX1, all the on-target
is pretty good for even
the literature mutants.
All of the literature
mutants and the Alt-R HiFi
reduced off-targeted editing
to detectable levels.
Very good.
Looking at HEKSite4,
where the literature
HiFis showed trouble on-target,
the Alt-R HiFi maintains.
And importantly, the
off-target editing
is reduced for all proteins
down to an undetectable level.
And it's the same story
for the VEGFA3 locus.
On-target is maintained,
all three HiFis
reduce off-target to
an undetectable level.
And we had thought that
this is really good,
but we really need to look
at global off-target editing
and really challenge the system
and get a feel for how good is
this protein.
So to do so, we utilized
a two-step approach.
And the first step was to
use the GUIDE-Seq procedure
to identify what the
off-target sites were
to start with and then
design the amplicons
around those off-target
sites and use
multiplexed amplicon-based NGS
to actually do the sequencing
at every one of those sites.
So first, using
GUIDE-Seq to find out
what the sites are and then
multiplexed NGS to sequence
at every one of those sites.
And we tested several
different guides
and looked at the results.
And before I get into
that I'll show you
just what standard GUIDE-Seq
demonstrated, comparing Wild
Type to HiFi delivered as RNP.
And with Wild Type, if you
look on top, the first two
VEGFA3 and EMX1
experiments, there's
only around 35% on-target
for both these guides.
They have a lot of
off-target editing.
The IDT HiFi at the
bottom reduces that,
so that you're up
to 80-81% on-target.
Moving to the right, there
are two guides, GRHPR
and the HPRT 38285 site.
These guides, for
whatever reason,
are inherently low off-target.
So probably no HiFi needed.
But the following
three guides are
sort of an intermediate
level, where you've
got 79, 64, and 55% on-target.
And in every case the IDT HiFi
brings that up quite a bit.
And through a lot
of investigation
with the GUIDE-Seq
procedure, we've
come to appreciate that it is
very, very good at identifying
what an off-target site is.
It's very good at getting
you the total repertoire
of edited sites.
Where it begins
to fail, however,
is how quantitative it is when
assigning an indel frequency
to each locus.
That's why we went on to
using this two-step procedure.
So we took what we
knew about these sites
and we did the
amplicon-based NGS.
And we also upped the
ante a little bit.
We did both RNP delivery
of Wild Type and HiFi,
but we also created
stable cell lines
where we constitutively
expressed Wild Type
Cas9 or the HiFi mutant.
These are essentially
the same cell line,
except we introduced the HiFi
mutations into this Wild Type
background.
And in the log-based
graph here, you're
showing in this orange
color the on-target editing.
The far left is
the on-target site.
You can see that's quite
high through this guide.
And then of all
the blue sites are
evidence of off-target editing
in different frequencies,
all rank ordered.
So you can see for
stable cell line
delivery of Wild Type Cas9,
only 43% of the total editing
occurs on target.
And if we simply introduce
the HiFi mutations--
again this is a system where
we didn't develop the HiFi
Cas9 for this.
We developed it
to work with RNP.
But simply introducing
those mutations
brings 43% on-target up to 97%.
So quite an improvement there.
And then looking
at RNP delivery,
this is an example where using
the Wild Type Cas9 by RNP,
there are a handful of
persistent off-target sites
that you can't
seem to eliminate.
So you've got still 73% of
the editing is on-target,
and then delivering
the HiFi Cas9
by RNP brings that up to 99%.
Again, this is one guide.
This is the EMX1 guide.
But if we extrapolate that
to four different guides,
you can see it's
essentially the same story.
A lot of off-target editing
when we use Wild Type Cas9
with stable cell line delivery.
You can clean up a good deal
of that in stable cell line
context by introducing
the HiFi mutations.
Alternatively, if
you use RNP delivery,
you can use the Wild Type Cas9.
You still get some persistent
off-target edited sites
with certain guides, but
you do clean up a lot of it.
And then RNP delivery
of the HiFi Cas9
gets you the rest
of the way there.
And in every case we saw
99% or greater on-target.
So this is really a real example
of the utility of this protein.
And I think what
we've done here is
we've engineered something
that works so well
on-target that, even if you're
a little uncertain of how much
you need to worry about
off-target editing,
you can mitigate any risk by
using the HiFi Cas9 delivered
as RNP.
And we've received some
pretty great questions
when presenting at conferences
or posters at conferences.
And one that came up is you
guys have introduced mutations
into Cas9, but how do you
know that you haven't changed
the nature of the
cleaved products in a way
where using this mutant is going
to affect the repair profile?
So we actually took
a look at that.
And what you're seeing
here is one example
of a lot of different
guides that we've analyzed.
Looking at the indel
profile at the cut site,
comparing Wild Type to HiFi.
So in green here is cells only.
And because there's
no edited sites,
it's going to be 100% at zero.
There are a couple
insertions that are present.
And you can see that
for Wild Type and HiFi
the frequency is
basically the same.
And the same is true
for all the deletions
that occur at this site.
And taking it in the
aggregate, looking
at a lot of different
sites, we can conclusively
say that the HiFi mutant gives
basically the same repair
profile as Wild Type.
And you can use it without
fear that we're going
to alter your repair profile.
Very good question
though, and it
speaks to the level
of friendliness
we have with the academic
environment and how, you know,
we sort of have a
symbiotic relationship.
Another question that
has come up from that
is how well is the
HiFi protein going
to work for HDR experiments?
And this is another
set of experiments
from our collaborators at
Stanford, the Porteus Lab,
and they're trying to do
correction of mutations
of the betaglobin locus.
And what they're showing here
is they have successful HDR
with the Wild Type Cas9
protein, but they're also
showing that they're getting
simultaneous indel 3 formation
at this off-target site.
And they used our HiFi
protein, and they're
showing, again, good HDR
but significant reduction
in off-target indel formation.
But what they noticed
is a modest reduction
in on-target editing
efficiency with our protein.
And there are going to be
certain sites where the HiFi
Cas9 has on-target issues.
It's not a perfect system.
But what they were able to
do is to add a little bit
more protein and get the
editing levels back up
to that of Wild Type and
beyond without sacrificing
the reduction in
off-target editing.
So even in these cases
where you do see modestly
reduced on-target editing, you
can simply add more protein
and solve the
problem without fear
that you're going to introduce
more off-target editing events.
And this is something
that we're really
excited about because there are
some big labs, both the Porteus
Lab, Jacob Corn, and
a few others, that
intend to take this protein
actually into clinical trials.
So something we're very
proud and excited about.
And we're always asking new
questions and trying new things
and working to improve things.
So I'm actually very
excited to give you
some data showing you
the improvements we've
made to these proteins.
And we'll start with the Wild
Type Cas9 and where actually--
where we've actually increased
the editing efficiency
by making modifications to
the linkers and NLS sequences
that surround Cas9
delivered into cells.
And this is an
experiment where we've
done gene editing to 12
different loci within HPRT,
comparing our gen 1 commercial
Cas9 product, in blue,
to the version 3
product in orange.
And you can see at every
one of these sites,
we've increased editing
efficiencies dramatically.
This is a minimal dose.
This is a 400 nanomolar
dose of the RNP complex.
And we've also put
this series of changes
into the context of
our HiFi protein.
And what you're looking
at in this experiment
is both on-target
editing in blue
and then all the off-target
sites in various colors.
So we've compared our gen-1
and gen-3 Wild Type and HiFi
products for this experiment
for on- and off-target.
We've also looked at
various suppliers--
other suppliers
of Wild Type Cas9
to get an idea of where
our products performed
in the field.
And what you can see,
going from left to right,
is that our V3
Wild Type improves
on-target editing of the site
that was already pretty good.
Since we've improved overall
editing efficiencies,
it's not that surprising that
off-target cleavage goes up
a little bit.
We see that our
generation 1 HiFi
had a little bit of an
on-target problem at this site.
But moving on to our V3 protein
brings the on-target editing
levels even above where our
Wild Type V1 product was.
And importantly, we've still
maintained a strong reduction
in off-target editing.
So we've got something
now that works
better than our previous
Wild Type product but still
is very low off-target.
And if we compare that to
all these different suppliers
of Wild Type Cas9, you
can see that there's
a lot of variability.
There are some that
work sort of average,
some that work very
poorly, and then
a couple that work pretty well.
And what I would point out
is that our V3 HiFi product
is actually equal or better
than both of these options
while having much less
off-target editing efficiency
events.
So just to summarize
this section,
you can use RNP delivery
as a first measure
to mitigate off-target risk and
avoid immune system stimulation
toxicity that's experienced with
plasmid-based delivery methods.
You can use it to
mitigate off-target risk,
but there's always
the risk there are
going to be persistent sites.
And we're recommending
that you use the Alt-R HiFi
protein to sort of
clean up the rest
of these off-target
cleavage events.
And then finally, we've
got this improved context
for the Cas9 protein
that we've introduced
into every one of
our Cas9 products.
And that begins, obviously,
with the Wild Type product.
We previously offered 100
and 500 microgram quantities.
We're now selling at
larger half mig quantity.
We also have this in
the context of our HiFi.
Again, we've introduced
now a half mig quantity.
We did not previously
sell a dead Cas9 protein,
but now we are going
to have this available.
And one thing I think
is unique about this
is we have quality controlled
this not only to be
dead for nuclease
activity, but we also
confirmed that it retains
RNA guide DNA-binding
activities to make sure that you
get an active and dead protein.
And finally, we have our D10A
and H840A nickase proteins.
We've upgraded those as well
to put them in that V3 context.
These are to produce
targeted cleavage
of just a single strand.
And I direct you to our
previous IDT webinar
on the use of these products.
And these will be available by
the end of the month, actually.
So these will be a replacement
on our website for the V1
products.
So I want to spend the
remainder of the webinar
giving a very brief summary
of the Cas12a or Cpf1 system
and then just
quickly demonstrating
the improvements we've made
in this protein as well.
This is another
RNA-guided endonuclease.
The one that we're selling is
from Acidaminococcus species.
Unlike Cas9, which exists
natively as a two-part system,
this is a single guide
RNA-facilitated system,
where you can use a 41-44 mer.
The double-stranded breaks
that are made with this system
leave staggered ends instead
of blunt cuts with Cas9.
And the PAM site is located
on the five prime end
of the target instead of
the three prime of Cas9.
And the PAM sequence is
actually a TTTV or any triple T
that is not followed
by another T.
So very, very different
from the NGG PAM.
And we've done an
awful lot of work--
and some of this is present in
a previous webinar demonstrating
length optimization of the guide
as well as certain chemical
modifications you can introduce.
And ultimately what we're
selling as a commercial product
is a 21 base-targeted site.
And you can also use RNP
delivery with Cas12a or Cpf1.
And it's basically
the same story
as Cas9, where DNA or
plasmid-based and mRNA delivery
methods have higher off-target
events than protein delivered
directly.
So we are recommended--
we recommend RNP delivery.
It's been reported that
Cas12a is intrinsically lower
off-target than Cas9.
We're a little
uncertain of that.
We wonder if Cas12a is just
a slightly lower activity
nuclease than Cas9.
It appears to be that
way, but the jury's
still out on that one.
And because this is
not a two-part system,
it's used as an RNP.
It's even simpler, where you
really only have two steps.
You can take purified or
commercially acquired Cpf1,
add it to your guide RNA,
form the RNP complex,
and then deliver that directly.
It's very, very simple and easy.
We have not had
consistent results
with lipofection
delivery of Cas12a,
so we do not recommend
using lipofection.
However, we do have great
protocols for electroporation
for this system.
And I think it's
important to note
that when doing
electroporation, we
find that this
system is actually
dependent on an enhancer
molecule that we sell.
And these are the results
demonstrating that fact.
In blue you have GNA
experiments with Cas12a
where we don't have enhancer.
And then in orange and
gray we have experiments
where we do have enhancer.
So we highly recommend
require using the enhancer
with the system to
get good results.
And I mentioned that this
is a trip TV PAM site, not
the previously
reported triple TN.
And I think that
these results nicely
demonstrate that fact, where
we look at editing efficiencies
at a lot of different
sites in this green color.
These are quadruple T PAM sites.
And you can see those are not--
the vast majority of those
are no editing detected at all.
Whereas for the other putative
PAMS, triple TA, triple TC,
and triple TG, those
appear to be much better.
So for that reason,
we don't think
people should select
quadruple T PAMS for Cas12a.
This is a summary of that data.
So in orange, this
is a ski slope plot
showing the editing
efficiencies of a lot
of different Cas9
sites that we tested.
So this system works well.
If we look at Cas12a where
we select any triple TN site,
you can see that we
end up throwing out
a lot of those sites because
they don't work at all.
And if we simply subtract
out the quadruple T PAMS
and only select
the others, you can
see that vastly
improves the system here
in gray, approaching the
same type of efficiency you
get with Cas9.
And we're hoping to
make that even better,
as we're testing greater than
1,000 sites for the production
of an algorithm
to better predict
good cleavage site for Cas12a.
Lastly, I want to show
you just very briefly
the type of performance
improvements
we've made with
this system, again
by introducing changes
into the Cas12a protein,
optimizing linkers, changing
MLSs, and moving things around.
Ultimately what we've settled
on is this V3 Cas12a protein.
And the performance improvements
seen with this system
are just absolutely phenomenal.
So looking at blue and orange,
this is our V1 Cas12a protein
builder with a variety of
different guides as RNP.
And then taking the same
guides and switching
to our V3 product, you can see
it's just an absolutely night
and day difference.
So we've really made significant
improvements with this system.
And then we've got the same data
plotted in a box and whiskers
plot on the
right-hand side here.
Again, blue and orange
are different replicates
of our V1 offering, and
then gray and yellow
are our V3 system.
So, again, this is our
V3 Cas12a, formerly known
as cpF1 protein,
and this V3 product
will be available at
the end of the month.
So very, very,
very, very shortly.
So what should we take
home from this talk?
Well, RNP delivery of Cas9- and
Cas12a-based systems, I think,
are the way to go.
It simplifies everything.
You eliminate the complexity
from immune system stimulation
and toxicity, and
you can dramatically
reduce off-target editing
without using high-fidelity
proteins.
For Cas9, I think
you can comfortably
use the HiFi protein
to ensure that you're
going to get good
on-target editing
and exceptional reduction
of off-target editing.
We've improved that even
further by putting it
in the context of
our V3 proteins.
We are getting
exceptional performance,
and you're dramatically
increasing editing efficiency
in live cells.
And for the context
of HiFi, you're
not doing that at the expense
of reducing off-target editing.
And again, all of
these things will
be available on
our CRISPR website
at the end of the month.
So with that, I'd like to
thank everybody for attending
and I'd be happy to
take any questions.
Thank you, Chris, for that
very informative presentation.
We will take the
remainder of the hour
to answer any questions
that have come in.
The first question from
Kelly is, "Do shortened
guide RNAs work as well
with IDT's HiFi Cas9?"
Yeah, so we don't recommend
using short guides
with Wild Type or
HiFi, because it
seems to be very context- and
site-dependant, whether they
work or whether they don't work.
And I think the HiFi is slightly
more susceptible than the Wild
Type.
But in general I
wouldn't recommend using
shortened guides with either.
Thank you.
Next, from Steven, "How does
HiFi Cas9 compare to the Hypa
Cas9, with on- and
off-target activities
when delivered as RNPs?"
OK, so yeah, we've look at--
we've actually looked
at a lot of these.
We looked at Hypa, we've
looked at the newer Evo Cas9,
and we've also looked at XCas9.
And what we've
found is that it's
sort of the same story, where
they're very, very, very good
at reducing off-target effects.
But when we test them as
RNP, the on-target editing
really suffers.
And I would say that
Hypa, Evo, and XCas9 all
produce lower on-target editing
efficiencies than the two
that I showed today, the
eSpCas9 and the eSpCas9 gen 1.
OK, next William
would like to know,
"How valuable are programs,
including IDT's, that
predict the guide
RNA efficiency?"
I think they're
extremely valuable.
I just think it's
such a difficult thing
to do that, you
know, eventually--
I think eventually someone
is going to get it right
and it's going to
really improve things.
But, you know, I just
don't know how much
work it's going to
take to get there.
I really think it's turned out
to be something that's really,
really challenging to do.
So to answer your question,
I think it's invaluable.
Right.
Next, Felix would
like to know why
you focused on using GUIDE-Seq
rather than CIRCLE-Seq
in the presentation.
I think that, you know, there
are many ways to do this.
And, you know, each has its
advantages and disadvantages.
And GUIDE-Seq was one of
the first to come out,
and we latched
onto it because it
allowed us to analyze local
off-target editing in living
cells.
And, for that
reason, we chose to--
because there's a
lot of development
that goes to even
reproducing things
that happen in the literature.
So we put our effort
into GUIDE-Seq,
and we found what
worked for us internally
and we just kind
of stuck with it
and incorporated it
into what we thought
is the best overall approach
to look at off-target editing.
So, you know, I think any
of those different sites
CIRCLE-Seq, Digenome-Seq, what
have you, I think they're all,
you know, they can all be used
and produce similar results.
Great.
Thank you.
We've gotten a couple
variants of this question--
what changes were made
between V1 and V3?
Were they adding more
mutations or optimizing
expression or purification
protocols or MLS and et cetera?
OK, so what I can
say about those
is we did not make any
additional substitutions
to the native [INAUDIBLE].
These are changes to the
regions flanking tags, linkers,
and nuclear
localization signals.
Great.
Thank you, Chris.
Elaine would like
to know if we have
noticed any change in Cas12a
V3's off-target profile.
That is a really,
really great question,
and we're just going
to have to look at it.
It's something we haven't
looked at very closely yet.
But no, it's a great question.
You know, I think
we were so blown
away by what we
made that, you know,
we hadn't sort of
gotten to that yet.
But it's a great question,
something we will address.
Yes, absolutely.
We have a question asking if
we have tested the immune cell
activation with RNP.
You showed at the beginning
the activation with IDTs.
But we haven't seen
any increased activity
when using RNP at all with
immune cell activation,
I think is what this
question is getting at.
OK.
Next, Theodore would
like to know if there's
any indication if CNR--
the crRNA length can alter
the cut site position.
That's an interesting question.
You know, that's just not
something we specifically
looked at, but I know that
we've shortened the crRNA,
and we're able to look
at repair profiles
and all that sort of stuff
for any of these experiments.
And you would expect that the
indel profile will probably
change if you're going to
change the cut site position,
and we've seen no
evidence of that.
All we really see is a change
of frequency indel formation.
We haven't seen any--
and of the changes
we've made, we
haven't seen any difference
in the cut products
or the frequency
in repair outcomes.
Great.
Thank you.
Liam would like
to know if the RNP
system is effective for
modifying bacterial genomes.
Yes, so the answer to that
question is I don't know.
I do know that bacteria have
extremely powerful three
to five [INAUDIBLE] nucleases.
And, you know, I also know that
it's not a super common thing
to try and deliver a protein
directly into bacteria
via electroporation
or chemical methods.
I do know there are
reports of people getting
fluorescent proteins in.
I've never seen
anybody get an RNP in.
That's something we've talked
about trying to get to work.
But no, I just don't
know the answer to it.
I don't know if
anybody's done it yet.
Right.
We have a question if
we are doing any work
to reduce the size
of the Cas9 protein.
The answer to that question
is we're not doing anything
to reduce the size of SpCas9.
I don't know that it's
extremely modular.
I don't know what we could--
what we could give
up and mix and match
without completely
disrupting its function
or making unintended changes.
But, you know, we're always
looking into new systems,
and I think if
somebody is to find
a similar Cas9 from perhaps
different species that
is smaller and gives the
same quality of editing
efficiencies, that
would probably be ideal.
But no, it's not something
we're actively working on.
Thanks Chris.
We've got a few more
questions that are coming in.
Natalie would like to know if
it's OK to increase the Alt-R
Cas9 concentration to
increase efficiency--
so increasing the
RNP concentration
to drive up your activity.
Yeah, I mean, I
think absolutely.
I mean, it just depends on--
I mean, every system is going to
have limits in terms of what is
and what is not toxic.
But I think it's definitely
a reasonable thing.
In fact, every time
in the laboratory
we look at a new
guide, typically
we'll do a dose response
going from high to low,
looking at both
on- and off-target.
So I think it's totally
a reasonable thing
to add more and try
to stress your system
and find out where your
particular guides and RNP
complexes are toxic or not.
Great.
Axel would like to know if you
could provide any advice on how
to optimize the
Cas9 complex for HDR
rather than non-homologous
end joining.
Yeah.
So Ashley, do you know if we
have the app note online yet,
for HDR?
We do.
So Chris is
referencing an app note
that you can access
on our support link
on our website that's going to
provide some basic information
for delivering a short,
single-stranded oligo, which
is an Ultramer for HDR that
shows high activity with RNP.
Yeah.
So there are a lot of questions.
HDR is obviously
more complicated.
Do you use enhancer?
Do you not use an enhancer?
What concentrations do you use?
And I think the
best thing I can do
is direct you to that
app note on our website.
Yes.
And we are actively working on
ways to improve HDR as well.
And we'll have more information
on that as it comes out.
Absolutely.
Dale would like to
know if IDT's HiFi
Cas9 can tolerate a heterologous
G at the beginning of the guide
RNA.
Heterologous G at the
beginning of the guide RNA.
So I guess if you're using
an in vitro transcribed sgRNA
and having a non-templated G?
Correct.
I mean, I think
it's going to be--
I wouldn't recommend doing it.
I think it's better to use the
chemically synthesized system
where you know what
you're putting into cells.
But you could empirically
test it and try.
I just think it's going to be
kind of a mixed bag of what
sites work and what don't.
And the same is going to be true
for wild-type in some cases.
So I think I would recommend
using a chemically synthesized
two-part system or
single guide RNA, where
you're not introducing
non-templated nucleotides.
Great.
Thank you.
Another question
that has come in
is if the V3 proteins still
require using the Alt-R
Electroporation Enhancers.
Yeah.
That's a really great question.
I think there are still
a handful of sites,
especially with Cas12a, where
the overall editing is low
and you still see a
pretty significant jump
with the enhancer.
So for Cas12a specifically,
I think the answer
to that is yes.
It's still something that
should be put into practice.
It's certainly
not going to hurt.
For Cas9, we're saying
it's not a requirement
but we still recommend using it.
And I think, on
the outside chance
that you've picked a guide that
is going to give poor editing
efficiencies regardless of
what protein you're using,
it's still not a bad
idea to use the enhancer.
It's not super expensive
and I don't think
it's going to hurt at all.
So I think the answer
to that is yes.
We're still
recommending using that.
Yes.
And all of the
other Alt-R products
will work seamlessly
with the V3 proteins.
Yep.
There was a question if we will
still offer the V1 proteins.
So they're going to not be
available on the website.
But we understand that there are
certain people that are maybe
in the middle of an
experiment where they need
to use the exact same protein,
and if that's the case,
you can simply call
our support line
and we will have
limited quantities
available for those
customers for, again,
a limited period of time.
Great.
Christina has a question.
She's asking if you
can explain again
what the difference
between Cas9 and Cas12a is.
Maybe there's some confusion
with the new nomenclature
switch of Cpf1 to Cas12a,
but briefly describe that.
Yeah.
I kind of figured that I'm
giving this webinar as--
maybe it should
have stayed Cpf1.
But yeah.
So they are very, very
different enzymes.
Cas9, from a different bacterial
species, from strep pyogenes,
recognizes an
NGG-based PAM that's
located at the three-prime end.
It's natively targeted by two
different RNAs, whereas Cpf1
or Cas12a--
we use the version
from Acidaminococcus--
it targets an entirely
different triple-T VPAM
that's located on the five-prime
end of the targeted sequence
instead of the three-prime.
Even the nature of the cuts
is different between the two
enzymes.
Cas9 cuts blunt whereas Cas12a,
or Cpf1, leaves staggered ends.
So they are two very different
RNA-guided DNA endonucleuses.
All right.
We're going to do one
more question from Dale,
who would like to know what
the shelf life of the HiFi Cas9
protein is.
Oh, great question.
So we've tested
this and we haven't
seen any difference between
our HiFi and our wild-type.
We've tested this in so
many different ways, looking
at 4 degrees, stored it in
minus 20 in liquid phase.
We've kept it at
minus 80, and I think
it still shows 100%
function out to a year.
I don't recall off
the top of my head
what the official
recommendation is,
but we don't find that HiFi is
any less stable than wild-type.
Great question.
OK.
That is all the time
we have for questions.
I want to thank all of you for
attending today's presentation.
I also would like to think
Chris for his informative
presentation, as well as Ashley
for conducting the Question
and Answer session.
This is one of a
series of webinars
will be presenting on CRISPR,
as well as other topics.
We will email you about
these future webinars
as they are scheduled.
Also, as a reminder, a recording
of this webinar will be posted
shortly on our web site and
at youtube.com/idtdnabio.
There you will find several
other educational webinars
on such topics as genome
editing, genotyping,
next generation
sequencing, qPCR,
and general molecular biology.
Also, the presentation slides
for today's webinar have been
published on our SlideShare
page at slideshare.net/idtdna.
Thank you again for attending,
and we wish you the best
of success in your research.
