Articles, Blog

Increasing genome editing efficiency and specificity with optimized CRISPR-Cas9 guide RNAs

December 6, 2019


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
expand the presentation slide window for easier viewing. 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 to record
your presentation on our YouTube and Vimeo channels. 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.

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1 Comment

  • Reply Eming Rau November 14, 2019 at 1:02 pm

    43:03 a nice table

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