Hello, and welcome to this
Integrated DNA Technologies
webinar, Increasing
Genome Editing
Efficiency and Specificity with
Optimized CRISPR-Cas9 Guide
RNAs.
My name is Sean McCall.
And I will be
serving as moderator
for today's presentation.
Today's presentation will
be given by Ashley Jacobi.
Ashley is a senior
staff scientist
in the Molecular Genetics
Research and Development Group
at IDT.
Ashley has been with
IDT for 12 years.
And during that
time, she has been
an author on 18
manuscripts published
in peer-reviewed journals,
contributed to numerous patent
applications, and has
presented in a wide variety
of international biomedical
research conferences.
Her current research
initiatives focus
on the development of
novel CRISPR RNA sequences
and modification
patterns that allow
for more efficient
and specific cleavage
by the SpCas9 and AS
Cas12a CRISPR nucleuses.
Ashley's presentation should
last about 30 minutes.
And following the
presentation, she
will answer as many questions
as possible from attendees.
The question and
answer session will
be conducted by Mollie
Schubert, research scientist
in the CRISPR Group here 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 entering your question in
the question and answers box.
Also, please note that you can
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In case you need to
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We will also post to record
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You will receive links to
these in a follow-up email.
So now let me hand it over to
Ashley for her presentation.
Thanks, Sean.
And good morning, everyone.
Today, I'm going to talk to
you about the different options
for introducing the
CRISPR-Cas9 guide RNA
and how to get the
highest efficiency
and specificity
for your systems.
And I'll start by giving a basic
overview of genome editing.
And then I will spend
a lot of time focusing
on the different
forms of guide RNAs,
specifically in-vitro
transcribed single-guide RNAs,
chemically synthesized
2-part guide RNAs, which
are comprised of a
cRNA and tracrRNA that
are in yield
together, chemically
synthesized single-guide RNAs.
I will also discuss the
benefit of introducing chemical
modifications into
your guide RNA
and give you a roadmap for when
to use which form of guide RNA.
I will also spend a bit of time
discussing off-target effects
and if there are any
differences associated
with the form of guide
RNA or Cas9 used.
And lastly, I will briefly
introduce our newest Alt-R
CRISPR reagent.
The two most common
RNA-guided endonucleases
that are used to edit
genomes in living cells
are Cas9 and Cas12a,
otherwise known as Cpf1.
Now on the left in this
slide is the Cas9 system,
where the guide RNA that
associates with Cas9
is natively comprised
of two RNA molecules.
In green, the cRNA,
which contains
the target-specific region shown
with a thicker bar, typically
about 20 nucleotides
in length, anneals
to the tracrRNA in orange,
which is the universal sequence
that associates with the cRNA.
The active complex
is then guided
to this target-specific area in
the genome upstream of the PAM
site, which is an NGG, and a
blunt double-stranded break
is made.
Now Cas12a on the right
side of the screen only
has a single short cRNA,
also shown in green,
which contains a 21 to 24
target-specific region shown
in the thicker bar.
It's also guided to
the area in the genome
now downstream of the PAM
site, which is a TTTV.
And in this case, a staggered
double-stranded break is made.
Today, I will focus on
the CRISPR-Cas9 system.
To implement CRISPR-Cas9
genome editing,
you need to introduce Cas9 and
the guide RNA to your system.
There are many ways to
introduce these components
into your system.
Now starting on the left in the
upper corner, Cas9 protein--
you can purify or purchase Cas9
protein to deliver directly.
Now moving down, you
can also make or acquire
a Cas9 cell line,
where Cas9 protein is
expressed all of the time.
You can purchase or
express Cas9 mRNA
that can be transfected directly
into your cells, as well.
And you can also deliver
a Cas9 expression
cassette that will be
transcribed and translated
in the cell.
Now very similar story
for introducing the RNAs--
moving over to the right side
of the screen in the top right
corner, you can deliver
chemically-synthesized guide
RNAs, which have the
advantage of adding
chemical modifications.
These can be synthesized,
as I mentioned,
in a 2-part form, where
the cRNA and tracrRNA are
in yield together or
can be synthesized
as a single-guide RNA,
where the two components are
fused with a linker loop.
You can also in-vitro transcribe
the single-guide RNA from a DNA
template and then deliver this.
Or you can deliver a
DNA expression cassette
as a linear DNA fragment
or as a plasmid.
And of course, you could
also combine the Cas9
and guide RNA into one large
plasmid, and deliver that.
At IDT, we favor direct delivery
of the ribonucleoprotein
complex, where we
introduce Cas9 protein--
we incubate Cas9 protein to send
synthetic guide RNAs in a test
tube and then directly deliver
that preformed complex.
One reason we favor delivering
the Cas9 and the guide
RNA as an RNP complex is
because it allows for very
fast and efficient delivery.
Now I'd like to go into
more detail on how easy it
is to form the RNP complex.
First, if you are
using a 2-part system,
you have the cost
effective advantage
of just synthesizing the
short RNA component, the cRNA,
every time you're
looking at a new target,
as the tracrRNA is universal.
But you do need to anneal
these two strands together.
We do this simply in step 1
by adding them an equal molar
amounts, heating to 95
degrees for five minutes,
and then slowly cooling
to room temperature
for about 10 minutes.
And then in step 2, we
incubate the active guide
RNA complex with
purified Cas9 protein
at at least a 1-to-1 molar ratio
for about 10 to 20 minutes.
And then we are ready to
directly deliver the RNP
complex into your system.
We have many protocols
on our website for how
to deliver the RNP complex.
We have detailed
protocols showing
how to do this via
lipofection, electroporation.
And we also have many
user-provided protocols
for micro-injection
or more niche systems.
The RNP complex
can also easily be
generated using a
chemically-synthesized or
in-vitro transcribed
single-guide
RNA where the cRNA and tracrRNA
are already fused together.
The method is the same.
However, there is no need to
preanneal the cRNA and tracrRNA
together, as they have
been synthesized together.
Otherwise, you incubate
your single-guide RNA
with your Cas9 name protein
for 10 to 20 minutes,
and you're ready to
directly deliver this.
Now I would like to discuss the
different forms of guide RNAs
and improvements
we've specifically
made to the
chemically-synthesized versions
that we offer at IDT.
Initially, a common way
to introduce the guide RNA
component was by
in-vitro transcribing
this single-guide RNA
from a DNA template
and then introducing
this to your cells.
This can result in
successful editing.
However, it is often accompanied
with large-scale cell death.
And here, in the
far left panel, I'm
showing HEK-293 cells that
constitutively express Cas9.
But there has been no treatment
administered to these cells.
These cells have been
allowed to grow up
to complacency over 48 hours.
The middle panel
has 30 nanomolar
of an in-vitro transcribed
single-guide RNA transfected
into these cells, while
the right panel has
30 nanomolar of a
chemically-synthesized and
chemically-modified
guide RNA delivered.
And you can see in
the middle panel,
the delivery of the in-vitro
transcribed single-guide RNA
was quite toxic.
And the cells have undergone
a lack of cell proliferation.
However, the
chemically-synthesized and
modified guide RNA delivery has
not affected the cell growth
at all on the right panel.
Looking further into
this, we published a paper
earlier this year showing
that unmodified guide RNAs,
either in synthetic form or
in in-vitro transcribed form,
induce an interferon response.
Now here, we are comparing
four different guide RNA
forms delivered into human
peripheral blood mononuclear
cells.
Now if you draw your attention
to the table in the top right
corner, I'll go through the
four different RNAs that
are used in this experiment.
Sample A is a guide RNA that
was chemically synthesized
but no chemical modifications
included-- no nucleus
stabilizing the
chemical modifications.
This is an unmodified
chemically-synthesized RNA.
Sample B is also
chemically synthesized.
But we have introduced
chemical modifications,
such as 2-prime O-methyl
bases and phosphoro phthalate
linkages to stabilize this RNA.
Now sample 3 is an in-vitro
transcribed single-guide RNA
that still has the 5-prime
triphosphate intact.
And sample D is also an in-vitro
transcribed single-guide RNA.
But we have phosphatase
treated this to remove
that 5-prime triphosphate.
Now to walk through a section
of data from this work,
please draw your attention to
the left area of the screen
where we have the black bars.
Here we are looking at
total editing of the four
different guide RNAs
we are looking at
compared to a positive control.
And what you can see here
is all four versions have
identical total editing
in this cell type,
whether we are delivering these
as in-vitro transcribed RNAs
or as
chemically-synthesized guide
RNAs with or without
chemical modifications.
So in this case, we
see identical editing.
However, now if you move
to the second figure,
we are directly
comparing here sample A,
which is a
chemically-synthesized guide
RNA with no chemical
modifications,
to sample B, which is a
chemically-synthesized guide
RNA containing
chemical modifications.
And what you can see is that
sample A, the unmodified RNA,
has induced significant
levels of interferon alpha,
while the RNA that has
chemical modifications included
in the guide RNA has not induced
any level of interferon alpha.
Now moving to the third panel,
we are now comparing sample A--
which is, again, an unmodified
chemically-synthesized guide
RNA--
now to an in-vitro
transcribed single-guide RNA
that contains the
5-prime triphosphate.
And now you can see that the
single-guide RNA, which is also
unmodified, has also induced
interferon alpha levels
and to a slightly higher degree
than the chemically-synthesized
form.
Now in the last
panel on the right,
we are comparing the
in-vitro transcribed guide
RNA that contains the 5-prime
triphosphate to one that
has had that removed.
Now you can see removing the
5-prime triphosphate does
reduce the level of interferon
alpha to a significant degree.
However, there is still
detectable levels.
The only time no interferon
response was detected
was when we use a modified
chemically-synthesized guide
RNA.
Now as I just discussed,
a large benefit
of using chemically-synthesized
guide RNAs
is the ability to include
chemical modifications that
will reduce the
risk of triggering
the cells in the immune system.
But adding chemical
modifications
also provides improved
nucleus stability to the guide
RNA, which results in increased
efficiency of the editing.
Another benefit of using
chemically-synthesized guide
and earnings is
that you will have
high-quality experiment-ready
reagents without needing
to do any work in your lab.
As I mentioned, the
individual cRNA and tracrRNA
can be synthesized separately
and then annealed in your lab.
And this is the most
cost-effective option,
because the smaller
RNA, the cRNA,
contains the
target-specific region,
and can be synthesized
by the thousands,
and can even be used
for library screens.
This then, as I've
mentioned, needs
to be annealed to the
tracrRNA, which is universal.
And this can be ordered
in large quantity amounts
and stocked in your freezer.
If you do prefer to have the two
components synthesized together
as a single-guide RNA,
that is also an option.
In our lab, we have
looked at hundreds
of chemically-modified
RNA oligos
to determine which bases of
both the cRNA and the tracrRNA
regions can be chemically
modified without harming
the activity of the guide RNA.
So here, we are looking at on
the right-hand side the cRNA.
And the first portion shows
the target-specific region,
the 20-base protospacer
guide domain.
And then the right-hand side
is the 16-base binding domain,
which is universal.
And what we are
showing in red are
bases that are
allowed to be modified
with 2-prime O-methyl bases.
We've also looked at other
2-prime modified bases
without any loss in activity.
Now= if an arrow is
indicated above the base,
a larger arrow indicates a major
loss in function if we placed
a modified base
at this position,
where a minor loss was shown and
was more of a sequence-specific
effect if there
is a small arrow.
Now on the left is the tracrRNA.
And we also show here
which bases in red
are allowed to be chemically
modified without losing
any activity of the guide RNA--
and again, with
large arrows, bases
that need to remain
as a natural RNA base.
So we have taken what
we've learned there.
And we now have three
different options
of chemically-synthesized guide
RNAs that we offer at IDT.
The first option on the
left, the Alt-R 2 part,
is what we've been offering
for three years now.
This is comprised of a
2-part system, wherein
green is the cRNA, which
is moderately modified,
and the tracrRNA, which is
pretty heavily chemically
modified based on what we just
learned in the previous slide
that I showed you.
And all of the RNAs
we offer it IDT all
contain chemical modifications.
It's just in different degrees
of how high that modification
is.
And I'd like to walk
through now specifically
what these different
forms are and when you
would want to use each form.
So as I mentioned, on the
left our standard system,
which is what's been
offered forever,
has moderate chemical
modifications.
And this works great in systems
where Cas9 is already expressed
and also works really great
when delivered as an RNP
complex for most sites.
Now our new systems
in the middle
is the Alt-R XT 2-part system.
Now this is also a
2-part cRNA tracrRNA
that needs annealed together.
But now the cRNA has
an increased level
of chemical modifications.
More of the bases have these
chemical modifications,
these nuclei-stabilizing bases.
But the tracrRNA is the exact
same tracrRNA we had previously
been offering.
So if you already have
this tracrRNA in your lab
and you want to try out
the more modified cRNA,
this is the same tracrRNA
we've always been using.
It's just now annealed
to a more modified cRNA.
And then the far
panel on the right
is-- we now offer a RNA molecule
where the cRNA and tracrRNA
portions are fused together--
so a chemically-synthesized
single-guide RNA, which is
100 nucleotides in length,
requires no annealing, has
a moderately high level
of chemical modification.
So the two forms on the
right work well, also.
And the advantage of using these
are if you were co-delivering
these with Cas9 in
an expressed form--
so when the RNAs need
to remain in the cell
for longer while waiting
for Cas9 protein to be made.
And I'll walk through some
experiments that highlight
that in the next slides.
These also work
well with our RNP
and can have a
slight advantage over
the standard 2-part
system, which
only has a moderate level
of chemical modification,
if you're working in difficult
experimental conditions
with, for example, high
nucleus environments.
And I did want to point out
that all three of these options
have a three to five-day
turnaround time.
So you can get these
experiment-ready reagents
pretty rapidly to
use in your lab.
So now I want to
go into some data
where I compare the three
different forms of guide RNAs
that IDT offers.
And I want to look at this in
the context of the Cas9 source
that you're looking at.
So to do this, we have looked
at 12 different guide RNA sites
that target HPRT.
And in this slide, we
are delivering these
into HEK-293 cells,
the electroporation.
We are taking the
12 different guide
RNAs that were synthesized
as a standard 2-part,
as the more modified two
part, the two-part XT,
or as a single guide RNA.
And we have complexed each
of these to Cas9 protein
and delivered as an RNP
complex with the guide RNA
at a 1.2 to 1 ratio
with the Cas9 protein.
And we're also including the
Electroporation Enhancer,
which I will go into more
detail in the next slide.
We're also taking
all 12 of the guide
RNA sites synthesized into
three different guide RNA
any forms, the standard
2 part, the 2-part XT,
and the single-guide RNA
and co-delivered these
with Cas9 plasmid or Cas9 mRNA.
And what we are looking at
is the presence of insertions
and deletions by NGS.
So now to look at the data,
if you draw your attention
to the top left
corner, we have now
delivered the 12 different
guide RNAs in the first section
as a standard 2-part.
And this is shown in
a violin plot, where
we've got the sum of
all the editing for all
of the sites shown.
And as the plot
gets wider, that's
where you have more of those
levels of editing represented.
And the white dot shows the
mean editing of all the sites.
But what's important to
see in the top left corner
is that all three guide RNA
forms have the white dot
at the same level of editing.
So you're not
getting any advantage
when you have delivered
these with Cas9 RNP,
delivering these
as an RNP complex
when you're using the more
modified guide RNA options.
Now moving to the
top right panel
where we are now delivering
this also as an RNP complex.
But we have dropped the
concentration 12-fold.
So the guide RNA is
now at 0.3 micromolar,
where it was initially
at 4.8 micromolar.
So going to a sub-optimal
level of delivery,
what you can see again now
is that the three guide RNA
forms have virtually the
same level of editing.
So you're not getting
any increased editing
here by using the more modified
2-part or the single-guide RNA.
However, now if you go to
the bottom left corner where
you are delivering Cas9
in an expressed form
as either a plasmid
on the bottom left
or as an mRNA on
the bottom right,
you do see a marked
improvement when
using the more modified 2-part
or the single-guide RNA.
And as I mentioned,
this is because the RNAs
are required to remain
in the cell for longer
while Cas9 is being
transcribed and translated.
So the more modifications render
these more nucleus protected.
And in these cases,
you would want
to use these more
modified versions
to achieve higher editing.
Now here, we are looking at
virtually the same experiment.
However, in orange, I have now
included another cell type.
We are now looking at
K562 suspension cells.
And this has been introduced and
the left panels the top right,
where this is delivered
as an RNP complex,
and in the bottom left, where
this was co-delivered with Cas9
plasmid.
And what you can see, again,
is if the three different guide
RNA forms were delivered
into, in this case, K562 cells
as an RNP complex, there
is no increase in editing
by using the more modified or
the single-guide RNA fusion
forms.
But in the bottom
left again, which
is delivering these
guide RNAs co-delivering
with Cas9 plasmid,
we see an advantage
of at using the
more modified forms.
So everything I just showed
you was in standard cell types,
HEK-293 and K562 cells.
Next, we wanted to
look at the efficiency
of the different guide RNA
forms and CD34-positive cells.
And we did this in collaboration
with Ayal Hendels Lab
at Bar-Ilan University,
where their goal
is to efficiently knock
out genes associated
with severe combined
immunodeficiencies
and to identify the best
guide RNA form to do so
with delivering these
as an RNP complex.
So the methods here
involve electroporating
into CD34-positive hematopoietic
stem and progenitor cells using
the Alt-R Cas9 nucleus
with in-vitro transcribed
single-guide RNA--
so, unmodified--
or with the standard
2-part system--
more moderately modified-- or
our new highly-modified 2-part
system, the Alt-R XT system, and
also with a single-guide RNA.
All of these RNP
complexes are being
tested with and without our
Alt-R Cas9 Electroporation
Enhancer.
And what this is is 100
nucleotide single-stranded DNA
that is non-homologous to
human, mouse, and rat genomes
but increases the
electroporation efficiency
of the RNP complexes.
One other thing they are
looking at in these experiments
is titrating the
amount of RNP delivered
to find the lowest level
to achieve the highest
editing in these primary cells.
So the first target they
are looking at here is RAG2.
Now as I mentioned, they are
delivering the RNP complexes
in increasing doses.
So if you draw your attention
all the way to the right
where it says 4
micromolar, here we
are delivering the guide RNA
as either in-vitro transcribed
single-guide RNA, as
the standard 2-part,
as the more modified 2-part,
or as a single-guide RNA.
Now you can see, at the 4
micromolar dose, the high dose,
in the dark red bars, there
is little to no difference
in total editing, just like we
saw in the 293 and K562 cells.
As these are being
delivered as an RNP complex,
you do not see any
difference in editing levels
if you are using an unmodified
or more chemically-modified
versions.
And this is true
walking down all the way
to the left part of the graph.
When you are
delivering these at 0.5
micromolar at a suboptimal
dose, you, again,
still see very similar editing
across the different guide RNA
sites.
But what is also conveyed in
this figure in the lighter pink
bars is the presence and
absence of our Electroporation
Enhancer.
And what you can see
and the light pink bars
are when these RNP complexes
were electroporated
without the
single-stranded DNA present
or in the dark red bars when
the RNP complexes included
the Electroporation Enhancer.
And you can see how the
Electroporation Enhancer
significantly boost
editing levels
for all of the guide RNA forms
in the CD34-positive cells.
Now here, we are looking
at a second target, RAG1,
with the same
experimental conditions.
Now again, if you draw your
attention to the far right
of the graph, the highest
dose of our RNP delivered
in this case, which
was 8 micromolar,
you can now see that we see
much higher on-target editing
when we're using the more
modified guide RNA forms.
Both the 2-part XT and
the single-guide RNA
have increased
production of INDELs.
I showed in the previous
slide that all guide RNA forms
have similar on-targeting
editing levels as these
are delivered with RNP.
And this set is also
delivered with RNP.
But it is worth
pointing out that we
do see a small subset
of sites, especially
when we're working in these
higher nucleus environments,
that additional chemical
modification can
be helpful to achieve
higher editing,
even if delivering with RNP.
This happens to be
one of those sites.
So if you have a site
that you are delivering
as an unmodified guide RNA or a
more moderately modified guide
RNA and you're not achieving
the level of editing
that you would like, it
may be helpful to use
one of these more
modified versions.
This slide also shows again
for this target, RAG1,
in dark green is the inclusion
of the Electroporation
Enhancer, while
in light green is
the absence of the
Electroporation Enhancer.
And again, you can see including
the Electroporation Enhancer
significantly boosts the total
editing for all guide RNA
forms.
So the first half of
my talk was focused
on looking at on-target
editing and levels
of the different
guide RNA forms.
Now I want to switch
gears slightly and look
at off-target analysis.
It is becoming increasingly
clear the importance
of assessing off-target effects
of your CRISPR-Cas9 components.
So specifically
here, we want to look
at if there are any
differences associated
with the form of
guide RNA you use,
as well as the form of Cas9.
It has been pretty
well published now
that off-target
effects are a concern,
and that delivering Cas9
at an expressed form
does increase the risk
of off-target effects
due to the continued and
long lasting of the Cas9,
and that delivering
Cas9 as an RNP
reduces off-target editing,
because the RNP is rapidly
degraded and doesn't
remain in the cell
as long as the expressed forms.
However, we have found that,
while our RNP does greatly
reduce off-target
effects, there are
some off-target sites that
do persist and are still
a problem.
So what do we do about that?
There's been a lot
of work done trying
to mitigate this-- and
work such as reducing
the length of the cRNA
to make it more specific,
introducing chemically-modified
bases at certain positions
to make things more specific.
And while these works
sometimes, they also
do reduce on-target editing.
And then there's also
been a lot of work
in looking at making
mutant forms of Cas9
that have higher fidelity.
There've been many great
publications discussing
various high-fidelity
mutants that were developed
through rational design.
But these were evolved
under conditions were Cas9
and the guide RNA were
expressed as plasmid.
And we have found when testing
these forums that delivering
these in RNP form does
reduce off-target effects,
but we also see a
significant reduction
in the on-target activity.
So we sought out to
develop a protein that
would maintain on-target
activity while reducing
off-target activity,
specifically when
we deliver these
as an RNP complex.
And I'm not going to go into
a great deal of detail here.
But we did this by using
a dual screening approach,
where we selected for
maintenance of an on-target
activity and absence of
off-target activity--
where we have a high-copy
plasmid on the left
that expresses a bacterial
toxin on the on-target site.
So this needs to be
cleaved to survive.
And on the left, we
have a second plasmid
that has an off-target site that
expresses an antibiotic marker.
And we need the
plasmid to not cleave.
So we made a random
mutagenesis library of Cas9,
and passed it
through this screen,
and assessed our positive hits.
And the mutant we
ended up settling on
is discussed in our "Nature
Medicine" manuscript that
was published last month, where
we show that we've identified
a high-fidelity Cas9 mutant
that, when delivered as an RNP
complex, maintains
on-target activity
but also reduces
off-target activity.
And we show this in
standard cell types,
as well as in human
hematopoietic stem cells.
And this is referred to
commercially as the Alt-R HiFi
Cas9 and is available
through IDT.
If you want any more
detail about that protein,
this is the publication
that highlights all of that.
I did want to show one key
figure from this paper just
to show how this
version does maintain
on-target activity when
delivered as an RNP complex.
So what we are looking at here
are 12 different guide RNA
sites.
And we are looking at just
total on-target activity here.
And we are comparing this
to on-target activity
of a wild type Cas9, again,
delivered as an RNP complex.
So you see those levels in blue.
And then if you go to the
end of each set, in green
is the Alt-R HiFi Cas9.
And so what you can see as you
walk across the graph is that
the new IDT HiFi Cas9 has
very similar on-target editing
levels when delivered as an
RNP as a wild type Cas9, while,
if you look at the other forms--
the other published
versions that
were evolved for Cas9 plasmid--
there is a market hit
in on-target activity
in the orange, gray,
and yellow bars
from the majority of the sites.
We know of target
effects can be an issue.
And we've evolved a new
protein to help mitigate this.
But we wanted to now look at
if the different guide RNA
forms have any difference on
the level of off-target effects.
So the next set of data
I'm going to show you
is looking at the most
common forms of guide RNAs
and assessing if these
have any difference
on the levels of off-target
effects that we detect.
So if you look at this
table on the top line,
we will be looking at a
plasmid single-guide RNA--
so delivering the guide
RNA in an expressed form.
And the next three options are
chemically-synthesized 2-part
guide RNAs, either as
completely unmodified--
as I've talked about in great
detail, our standard 2-part--
which is moderately modified,
and then the fourth line down,
the 2-part XT, which
has increased levels
of chemical modification.
Now the last three
lines in this table
are guide RNAs that
are synthesized it
as a single-guide RNA,
as one RNA molecule.
So the first of those three
lines in in-vitro transcribed
single-guide RNA, which is made
through enzymatic synthesis
but still has the
triphosphate intact--
and then comparing this
to chemically-synthesized
single-guide RNAs
that are unmodified
or the IDT
chemically-modified version.
Now before we show
the data, I do
want to take a step back
and discuss how to identify
potential off-target effects.
Which sites should
you be looking for?
There are many methods out
there to predict or validate
off-target sites.
And there are a lot of in
silico prediction tools, also.
Currently, a lot of
these in silico tools
do actually miss a
lot of important sites
and can also over-predict sites.
So it makes it a challenge to--
if you're going to do
amplicon sequencing
and assess all of these
off-target sites--
to know which ones to look at
and to have a manageable level
to actually study.
There have also
been publications
of different in-vitro assays
to define off-target effects.
But this is not unique
to your cell type
and can also often
be over-predictive.
The GUIDEseq approach
is commonly used.
And this is where
you empirically
determine off-target
sites in your cell type
through an unbiased approach.
But this is more of an
identifier and not overly
quantitative.
And at IDT, we have developed
the rthAmpSeq system for CRISPR
that we use in-house.
But this is not yet
commercially available.
And what we use this for
is, after identifying
what potential off-target
sites you have via GUIDE seq,
we now go and do a multiplex
amplification-based targeted
enrichment approach,
where we can
look at all of the
off-target sites
that we've identified through
either in silica prediction
tools or unbiased methods
and actually quantitative
the level of off-target
effects we've
seen in our delivery systems.
So we are able to
include an on-target site
and up to 1,000 off-target sites
in a single multiplex reaction.
And this is known as rthAmpSeq
seek for CRISPR, which
we currently use internally.
And this will be commercially
available later this year.
So the two sites we are
going to be studying
and the comparison of
the off-target effects
for the guide electroporation
are AR and EMX1.
So first, we need to determine
the potential off-target sites
that we want to look at
for these two targets.
So I just wanted to
highlight the workflow
that we follow in our lab.
So the first thing we do is
we use the GUIDEseq approach,
where we deliver our guide RNA
into cells that express Cas9
along with the GUIDE
seq double-stranded tag,
and identify where the
double-stranded tag is
being integrated to identify
potential off-target sites.
As I mentioned, we
are delivering news
into cells that express Cas9.
And we do this, because,
as I've mentioned,
delivering Cas9 in
an expressed form
has higher off-target effects.
So in this case,
we actually want
to go into cells that
have Cas9 expressed
to be able to cast a
wider net and assess
any potential risk that
we have for sites that
could cause mischief and have
an off-target effect happening.
Because of this, we also use
our more modified 2-part system,
again, because now this RNA
is going to be more stable.
So this basically just
gives us the highest chance
of finding all potential
off-target sites.
So the panel on the right
shows a typical readout
we will get after doing GUIDEseq
through the NTS analysis.
And the sites are all
identified that have
detectable reads
of the GUIDEseq tag
after making our
double-stranded break.
We also then do add
on into this list
some in silica
prediction tools that
have three to four mismatches.
And we take this list, and
we design and synthesize
a rthAmpSeq panel,
where we can then
multiplex all of these sites
together into one library prep.
So for the two sites
we'll be looking at today,
AR has 54 assays included in
it, the on-target assay and 53
off-target sites that we've
identified in this means.
And EMX1, which has 32 assays--
so the on-target assay
and 31 off-target
assays that we've assessed
through this workflow.
Now here, I do want to
show why we are deciding
to use our more modified
2-part system when
we do the GUIDEseq
identification approach.
And what you see here
in the top panel in blue
is when we have delivered
the 2-part XT guide RNA
into cells that express Cas9.
And we are assessing
off-target sites via GUIDEseq.
And every bar here
represents somewhere
where we have detected
an off-target site.
So the top panel is looking
at three different guide
RNAs in blue, where
we have delivered
a more modified 2-part.
And below this is
the same guide RNAs.
But in green is where we've
delivered a less modified
chemically-synthesized
guide RNA.
And what you can
see here is that we
are identifying more
sites when we're using
a more modified guide RNA--
again, because this guide RNA
is going to be more stable.
And the black numbers,
the percentages,
represent the sum of the total
unique reads that we're seeing.
So what this shows is
that the majority of the
reads that we are finding
for the two different guide
RNA forms had a high
level of overlap.
The more modified XT form just
is identifying more sites.
So again, this gives us
the greatest assurance
that we are now going to
use amplicon sequencing
and assess the highest
risk-- all potential risk
sites for off-target effects.
So now to move into the
experimental details of looking
at the off-target effects of
the different guide RNA forms,
we have transfected all of these
different guide RNAs variants
I discussed, targeting
AR and EMX1 into cells
that express Cas9.
And then we have also delivered
these as RNP complexes,
where we've taken the
different guide RNAs
and complexed these
to Cas9 nucleus
in either wild type or
the Alt-R HiFi form.
So now we are able to
look at the difference
in off-target effects of
the different guide RNAs
when delivered into
cells that express Cas9
or when delivered
as an RNP complex
in either wild type
or high fidelity form.
And any time we are
introducing an RNP complex,
we are including the Alt-R
Electroporation Enhancer.
And then we are assessing now
on and off-target editing levels
through the rthAmpSeq
system, where
we have panels defined based
on the previous work using
GUIDEseq to identify the
off-target sites for these two
targets, AR and EMX1.
Now there is a lot of
data in this slide,
so I'm going to try to
walk you through this
in a pretty detailed form.
So this first
target shows all of
those experimental
conditions targeting AR.
So if you look horizontally
across these graphs
in the things that are boxed off
with each individual pie chart,
these are the seven
different forms of guide RNA,
as a plasmid single-guide
RNA, as an unmodified,
and et cetera.
And now these seven
different guide RNAs names
are delivered
vertically into cells
that express Cas9 in panel A,
or delivered as an RNP complex
in panel B as a wild
type Cas9, or in panel C
with the high-fidelity Cas9.
Now if you draw your
attention to panel A,
I'd like to walk you through
what all of these bars mean.
So the first bar in orange
is the on-target assay.
So as I mentioned,
these panels are
designed to have
the on-target assay
and all of the off-target
assays included in them.
And in blue, I'm showing
the top 10 off-target sites.
We did run the full
panel, but this is just
showing the top 10 sites here.
And then the pie
chart shows you--
the focus here is to look at
the orange region and the number
there, because that's the
total reads that are on target.
So you can see, for example,
the single-guide RNA
at the far end of panel
A. The majority of those
reads actually are
off-target effect reads.
So there's a high level of
off-target editing associated
with this guide RNA site.
So if you look across panel
A and look at the numbers
above the orange bars,
what we're seeing here
is that we're seeing similar
on-target levels of editing
when we're delivering all
of these different guide
RNA forms.
And then if you look
at the blue bars,
you can see that all
of these guide RNAs,
when delivered
into Cas9 cells, do
have pretty significant
levels of off-target effects.
And if you look at the Alt-R
XT in the middle, which
has 34% of the
reads is on target--
so the majority of the
reads are off target.
Or if you look at
the single-guide RNA
at the end of that panel
A, these two forms,
which are the most modified,
do have slightly higher
off-target effect levels.
And this makes
sense, because these
are going to remain stable
in the cell for longer.
But across the board, delivering
all of these different guide
RNA forums into
Cas9 cells, there
are pretty significant
levels of off-target effects.
Now if we move down to panel B
and we look at the orange bars,
we can see that there
are similar levels
of on-target editing.
The unmodified
has slightly less.
And in this case,
the single-guide RNA
has slightly higher.
However, we've made a
market reduction now
in delivering these guide
RNAs as an RNP complex.
So as I mentioned
before, switching to RNP
is going to greatly reduce
your off-target effects.
But you can see, there are still
some blue bars that creep up.
But now the majority of the
reads that we're detecting
are 90% greater on target.
Any off-target sites are
below 1% of the total reads.
But they're still present.
So now if you move to panel C,
what's worth pointing out first
is that the numbers
above the orange bars
are virtually identical to
panel B. So as I mentioned,
we developed this
protein to maintain
high on-target activity.
And this nicely shows
how that is the case.
We are seeing very similar
on-target activities
when we are delivering
the high-fidelity
Cas9 as when we are delivering
the wild type RNP complex.
But now we have completely
removed all off-target effects
associated with any guide RNA
by using the Alt-R high-fidelity
nucleus.
Now the second target--
this is set up in
the exact same way--
is looking at EMX1.
Now this is a
popularly-published target
site, because it does actually
have pretty significant levels
of off targets.
And so now if you look
at panel A-- again,
we are delivering the different
guide RNAs into cells that
express Cas9--
you can see, again, very high
levels of off-target effects
associated with all
guide RNA forms.
And in this case, there's not
really a certain guide RNA
that stands out as having
more off-target effects.
They all have pretty
high levels when
you're using Cas9 in
an expressed form.
So now moving down
to panel B, again
take note of the numbers
above the orange bars where
we're seeing similar levels
of editing for all the guide
RNA sites.
So we're not biased there.
But now the blue bars
again-- now actually
switching to our RNP, there is
still pretty significant levels
of off-target effects.
60% to 70% of your
reads are on target.
But there is a good portion
that are actually off target.
So this is why I
mentioned that there
are some sites that
do persist, even
if you switch to RNP delivery.
So now, switching to
high-fidelity Cas9,
again, the level of
editing of the numbers
above the orange
bars are virtually
identical to the
wild type delivery.
But now we have virtually
reduced almost all
of the off-target edits
associated with this very
promiscuous guide RNA site.
And it's also worth pointing
out that all of the seven
different guide RNA
forms that we delivered,
as well as the three
different Cas9 forms,
produce identical
repair profiles.
So if you just
focus your attention
on panel B, what we
are looking at here
is the individual bars are these
seven different guide RNA forms
for this particular site, EMX1.
And the 0 mark is
anything that we sequenced
that was left at wild type.
So we saw about 85% editing for
the majority of these sites.
But then what we are
seeing is that this site
has a repair profile that
has a very predominant
1-base insertion and
then a 3-base deletion
or a 6-base deletion.
And what is really
interesting to see
is that all of the different
guide RNA forms had
the identical repair
profile in panel B.
And now if you look at
all the panels as a whole,
you can see not
only is the repair
profile the same for the
different guide RNA forms,
it's also the same for
the different Cas9 forms.
So now delivering these
into either Cas9 cells
or switching and using
our high-fidelity mutant,
we have not changed anything
with the repair profile.
So to wrap up the data
that I've showed you,
there are many different
guide RNA formats available.
And most of these give similar
on-target editing and levels
when they are delivered as RNP.
The higher
modifications, though,
do have a nice
advantage when you
are co-delivering the guide RNA
with Cas9 in an expressed form.
And there is a subset of
sequences, though, that
do respond better to
higher modification,
even if you're
delivering these as RNP.
And this does tend
to be a sequence
and cell-type dependent effect.
I went into great detail
about the benefits
of chemically-synthesized guide
RNAs having experiment-ready
reagents but also the ability to
add in chemical modifications,
which increase stability and
reduce the risk of triggering
the cells in the
immune response,
where unmodified guide RNAs
or in-vitro transcribed
single-guide RNAs induce
those high levels of--
things such as interferon alpha.
Then moving to the
off-target effects analysis,
I showed that the different
guide RNA forms really
result in similar
off-target editing levels.
There is a slight increase
with more modified versions.
But really, it's the
source of Cas9 that
drives the off-target editing.
So delivering the guide
RNAs as an RNP complex
does show a huge reduction
in non-specific editing.
But using the Alt-R
high-fidelity Cas9 really
further reduces any of
that off-target editing.
So I just wanted to
take a minute here
at the end and showcase
all of the different tools
we have now developed in
the research group at IDT
for nice CRISPR
tools that is now
a complete workflow,
essentially-- where,
if you look at the left panel,
we now offer a CRISPR-Cas9
design tool that has
pre-designed guides, custom
designs, and there's also where
you can check your existing
designs.
And this works for human, mouse,
rat, zebrafish and C. elegans.
And we will soon also be adding
and HDR design tool to this.
To make your cut, you need your
guide RNA and your proteins.
For the guide RNA, which we've
talked about in great detail
today, we have the 2-part system
as either the standard 2-part
or the more modified.
We have the
chemically-synthesized
single-guide RNA.
And then we also have
the cRNA for the Cas12a.
And as I mentioned,
all of our guide
RNAs all include
chemical modifications,
just the XT is more
modified than the standard.
We had a webinar
earlier this year
talking about the different
CRISPR proteins we have
and the improvements
we've made to increase
the efficiency of these.
Our current suite includes
the wild type Cas9,
high-fidelity Cas9,
both [INAUDIBLE]
versions and Cas12a.
And earlier this year, we
went from our V1 version
to V3, which--
the activity of these
is all now increased.
And as I talked about today,
we have the Electroporation
Enhancer, which increases
the efficiency when
you're delivering your RNP
complex of the electroporation.
And we have
Electroporation Enhancers
specific for Cas9 and Cas12a.
Now everything I
talked about today
was looking at the
non-homologous end-joining
repair, where we are just
specifically looking at INDELs.
We do have a suite
of options if you
are wanting to add in a donor
template to make a correction.
We have Ultramers, which are
single-stranded DNAs, that
go up to 200 bases.
And we also have Megamer
single-strand DNA fragments,
which can go up to 2,000 bases.
And what I'm going to talk
about lastly on the next slide
is our newest reagent,
the Alt-R HDR Enhancer.
And then lastly-- now you
have designed your experiment.
You have major
double-stranded break.
You have maybe introduced
a donor template
to make a repair.
Now to analyze your editing,
we offer the Genome Editing
Detection Kit, which is
a T7-based system, which
works great for simple
screening of your components.
But as I mentioned
today, we also
have developed in-house
the rthAmpSeq system
for CRISPR, which is a multiplex
amplification-based system
for Illumina 6 sequencing
that allows you to look
at up to 1,000 assays at once.
And this will be available
commercially later this year.
So I did just want
to end on talking
about our newest
reagent, because this
is really exciting.
We launched this last week.
We now have available
an HDR Enhancer,
which is a small
molecule compound that
increases HDR efficiency.
And here I'll just
show one slide
of data, where I'm showing that
we were able to increase HDR
rates with Cas9 and
Cas12a nucleuses
by including the HDR
Enhancer in the experiment.
So to the left of the
line, the first four sites,
what we are looking at is HDR
rates for four different sites
when the Cas9 nuclease.
So in blue, we are just
looking at HDR rates
of including the RNP complex
and a donor template.
But in orange, we have now
also included the HDR Enhancer.
And this is done in
Jurkat cells here.
But we've seen this
in many cell types.
And what you can see is that
the orange bars actually
increase HDR rates, in some
cases, by almost six-fold.
Now the same is
true on the left,
where we are now looking at
four sites that have a Cas12a pM
site.
So we are now delivering
RNP complexes for Cas12a
and including the HDR
Enhancer in orange
or just looking at
the natural HDR rates.
And again, you can see
here the marked improvement
of including this HDR Enhancer.
So this is now
available on our website
and is an exciting new
product that we have.
So to wrap everything
up, I just wanted
to put up a couple
take-home messages
about the different
guide RNA forms.
Because like we've
seen today, there
are a lot of different options.
So the standard 2-part
system, like I've said,
works very well for
many applications
and is the most cost-effective
synthetic option.
This works great
if you are working
with cells that express Cas9.
It works great as a tool
for screening guide RNAs
and even for looking
at gene libraries.
It works great with RNP delivery
the majority of the time.
And there have
actually been reports
that the 2-part system can
have greater efficiency
than single-guide RNAs in some
systems, such as zebrafish.
And then as I've
discussed today,
we have a 2-part system
that has increased chemical
modifications, the
Alt-R XT 2-part.
And we also have
the RNAs guide RNA.
And so these two options,
which have additional nucleus
stability, have an advantage
if you are co-delivering these
with a Cas9 plasmid or mRNA,
if you are delivering these
with lipid
nanoparticle delivery,
if you are working in
high nucleus environments.
And like I said,
there is a subset
of sequences that are more
susceptible to nucleus
degradation.
So adding more
chemical modifications
can sometimes really
improve your editing.
So that wraps up my
portion of the talk today.
And if you want any
more information,
we have a lot of really good
information on our website.
We have a lot of posters
and other webinars.
And we have a lot of
internal-generated protocols,
as well as protocols provided
by some of our users,
all available at the
website on the screen.
So with that, I want to thank
everyone for their attention
and pass it back over to Sean.
So, thank you.
Thank you, Ashley, for that
informative presentation.
If you have a question and
have not done so already,
please type it into the Q&A box.
We'll take about five to 10
minutes for questions now.
So here is the first one.
OK, so we had a few questions
about our Alt-R Electroporation
Enhancer that you
mentioned, Ashley.
Customers wanted to know how the
Electroporation Enhancer works,
what the mechanism of
action is, and then also
if we see any stimulation
of the immune system
when using the
Electroporation Enhancer?
Thanks, Mollie.
So the Electroporation
Enhancer is included--
it is not formed
with the RNP complex.
So we form the RNP complex.
And then we add
that to our cells.
And then we add the
electroporation Enhancer.
And that is all
electroporated together.
And what we see is
that this overall
increases the efficiency of
getting this into the cell.
Now the exact mechanism we are
still trying to understand.
But we see this as acting
as a career DNA, where
we are increasing the
shuttling of getting
these components into the cell.
And the Electroporation Enhancer
is a nice way to do this.
Another option is you can
increase the amount of guide
RNA in your RNP complex,
where you're now
adding an additional
nucleic acid in there.
But there really is a nice
additional boost in editing
when you include this.
And then the second part was?
What are the immune
responses, if any,
of the Electroporation Enhancer?
Yes.
We have not looked at that in
as great of detail as the RNA.
But the work we have done show
very little immune response
of this.
We have done a lot of work
to optimize the sequences
that we are using for these
DNA sequences, as well.
And we've also
looked into the rates
that this would integrate
into your genome.
And those rates are
also extremely low.
Great.
Thank you.
We had another few
questions about,
if you're able to use the RNP
complexes in other systems
such as fungi or plants--
if you have any
recommendations on that.
Yes.
There have been some
publications now
specifically with
plants that the RNP
system is very efficient with.
So we have definitely heard
positive feedback there.
I know there is work
done trying to understand
if this will work in bacteria.
And I haven't seen a lot of
publications on that yet.
But I know that's an area
people are actively working on.
Great.
And one researcher wanted to
know if the GUIDEseq results
that we showed in the HEK-293
Cas9 staple cell line--
do you think those
would be representative
of results in
other primary cells
that were expressing Cas9?
Yes.
And we have actually compared
with some collaborators Cas9
cells in different cell types.
And we do see very
similar levels
of which off-target sites are
seen across different cell
types.
Great.
Thank you.
OK, here we have a question
about that HDR Enhancer
that you just mentioned.
A researcher is
wanting to know how
it compares to other small
molecules tested, such as SCR7.
So before launching this, we
did a very thorough screen
of many different
small molecules.
And this is actually
the only one
that we saw that gave
this marked improvement.
We saw that it has a
much higher increase
in editing over any of the other
published molecules out there.
Great.
Do you have any data on human
embryonic stem cell editing?
For example, what
is the best combo
of reagents that could
be used for these cells?
We, actually, on our website,
do have some protocols
for that particular
cell type that I
would direct you to look at
specific conditions there.
But we have seen very
high levels of editing
with using the modified
chemically guide
RNAs complex to be a Cas9
protein, both the wild type
and the high fidelity.
Great.
Thank you.
These are great questions.
Thank you for
writing in, everyone.
Here's another one.
So do you observe any effects
of the RNA secondary structure
on the efficiency of editing?
That's a good question.
Considering the tracrRNA
and single-guide
RNA does have a lot of
secondary structure,
we have not noticed any
decrease in activity
with using the single-guide RNA.
This is as efficient
as the 2-part system.
Great.
OK.
And I think we have time
for just one last question.
We'll try to get back to any
other questions in the future.
But the final question
would be, do you
have modified guides
for the Cas12a system,
as well as the Cas9 system?
Yeah, so that's
a great question.
And as I mentioned very
briefly at the beginning,
this focus of this
presentation was Cas9.
We do have webinars available
on our website that specifically
discuss Cas12a.
But we do have the Cas12a cRNA.
So for Cas12a, it's just
a single short-guide RNA.
So the cRNA is about 40
nucleotides in length.
And we have studied,
again, hundreds
of different sequences with
various chemical modifications.
And what we offer
on our website is
chemically-modified Cas12a cRNA.
Thank you, Ashley.
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 thank Ashley
for an informative
presentation, as well
as Mollie for conducting the
question and answer session.
This is one of a
series of webinars
we'll 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 website and
at youtube.com/idtdnabio.
There you will find several
other educational webinars
on such topics as NextGeneration
sequencing, genotyping, qPCR,
and general molecular biology.
Thank you again for attending.
And we wish you the best of
success in your research.
