- Hi everyone.
So what I want to do is
tell you about a technology,
that grew out of a curiosity
driven research project
in my lab here at Berkeley.
And I'm gonna start by
just posing the question,
suppose we had a way to go into cells
and make a very precise
change to the DNA of the cell,
so precise that we could
change a single base pair
in the entire human genome, for example,
much like we might fix a
typo in a Word document?
Sounds like science fiction,
but this is actually
a technology that is now in our hands.
And it really came about
through not a focused effort
to discover such a
technology, but actually
through a curiosity-driven
research project,
that was aimed at figuring out
how bacteria fight the
flu, fight viral infection.
And for me, this project really started
with great conversation that I had
with a colleague here at
Berkeley, Jillian Banfield,
who is in the College of
Natural Resources at Berkeley,
and Jill does a lot of work with bacteria,
she likes to focus on bacteria
that have never been isolated,
never been cultured in the laboratory,
she's trying to understand
what's out there
in terms of the microbial world,
and in the course of her
research, she came across
a very interesting
observation, which was namely
that a lot of bacteria have
a very unusual sequence signature present
somewhere in the DNA genome of the cell,
that looks like this
where you have a series
of repeated DNA sequences
about 40 base pairs in length
and in between those
repeats are unique sequences
of also about 40 base pairs in length.
And people since 1987 had
occasionally noticed these
in their data, and these had
come to be known as CRISPRs
which stands for Clusters of
Regularly Interspaced Short
Palindromic Repeats, a big
mouthful, we like to use CRISPR,
and it refers to this
pattern of DNA sequences
that can occur in bacterial genomes.
And the reason that Jill
called me about this
was that we were working away
in my lab here at Berkeley
on ways that small RNA
molecules can control
the expression of genes,
much as what you just heard
about in Gary Ruvkun's talk.
And we were trying to understand
the molecular mechanisms
that underlie those pathways.
And Jill wondered whether
there might actually be
a role for RNA molecules
in a pathway that might stem
from the presence of these sequences
because in 2005 three labs had pointed out
that when they look these CRISPR loci,
they found that these unique sequences
in between the repeats,
very often corresponded
exactly to sequences that could be found
in the viruses that infect these bacteria.
And so together with that observation
and the fact that next
door to these sequences
are typically a set of
CRISPR-associator cast genes,
that covary with sequences over here,
the idea was maybe this is some way
that bacteria record
over time the infections
that they've experienced from viruses,
keep a little genetic
record in the context
of this kind of a sequence
pattern, and then somehow use
that sequence information
to protect the cells.
And Jill thought, wouldn't it be amazing
if that was happening through production
of RNA molecules, little
copies of these sequences
that might be made and
used by these cells.
And she knew we were working in RNA,
so she wondered if we would
like to test that idea.
And so, what emerged over
the next several years.
and one of the interesting
twists to this story,
is that the first genetic data experiments
that test this idea that
bacteria immune system
came from work done at a yogurt company,
Danesco by Rudolph Barrangou
and Phillipe Horvath,
was to show that in fact
bacteria that have a CRISPR locus
can defend themselves,
they can acquire immunity
to bacteriophage that
infect the cell by detecting
that foreign DNA as it
gets injected here shown
by a virus injecting this into a cell.
Little bits of that
foreign DNA are integrated
into the CRISPR locus in such a way
that they are stored there permanently,
they can be passed on
to the next generation
of bacteria, and then those
DNA sequences are copied
into RNA and form the basis
for targeted recognition
of sequences that have the same,
have a matching sequence
to what was recorded originally.
So these RNA molecules are broken up
into bits that each include
one sequence derived
from a virus, they assemble with proteins,
one or more proteins
encoded by the Cas genes
to form RNA protein targeting complexes
that use the sequence
information in the RNA
to base pair with foreign
DNA and allow these proteins
to cut up the viral DNA.
So this seemed like a
very interesting parallel,
actually to RNA interference,
in the way that micro-RNAs
work as Gary described
in the previous talk.
And so we thought it would
be really interesting
to figure out how
bacteria actually do this,
what's the mechanism for this
kind of a defense pathway.
And so in my lab, we started
working on this central part
of the pathway, namely how
these RNA molecules are created
in the cell, how they
assemble with proteins,
and how they carry out
this targeting mechanism.
And what emerged from that was a series
of mechanistic insights
that led us to eventually
partner with a colleague of mine,
Emmanuelle Charpentier who was at the time
located in Sweden, a
medical microbiologist,
and we decided to work together
to understand the molecular function
of a particular Cas protein called Cas9,
that is the only gene
necessary in some bacteria
for this viral defense
pathway to function.
And what emerged from
that collaboration was
that Cas9 is a very interesting protein
that is essentially programmable,
it can be programmed
with these bits of
CRISPR-derived RNA molecules.
And so by doing experiments
with purified proteins
and RNAs in our
laboratories, we figured out
that Cas9 has the ability to recognize
double-stranded DNA at sequences
that match a 20-nucleotide
sequence present
in the CRISPR RNA, which is this molecule
that's shown right here in this cartoon,
and at those sequences
the Cas9 protein opens
up the DNA double helix
positioning two active sites
in the protein to generate
a double-stranded break in the DNA.
Now there are a couple
of interesting twists
to the way this works.
We found out, first of all,
that this targeting has to occur
adjacent to a little motif
called the PAM sequence
in the DNA, so for this
particular Cas9 protein,
that's a GG-dinucleotide motif in the DNA,
or two GC base pairs next to each other.
And the other thing
was that this actually,
this system actually requires
a second RNA molecule
called tracer, which is this
red molecule right here,
which can interact by base pairing
with the end of the CRISPR RNA.
It forms the structure that
recruits the Cas9 protein.
So you really have to
have both of these RNAs
and the protein, and then
you can get this kind
of targeted double-stranded DNA cutting.
And so once Martin Jinek in my lab
had figured this out, he
realized that he could actually,
sort of like a good biochemist,
he was trimming away
at these RNAs and he
realized that he might
actually be able to
simplify the system relative
to what nature has done to
create a two-component system,
a single RNA that would
have the targeting ability
and the Cas9 capability
in the same molecule.
And so he created a
chimeric single guide form
of the RNA that has the
targeting information
in the molecule right here,
and it has the Cas9 binding
information over here.
And so this is an RNA
that can be programmed
by simply changing this
20-nucleotide sequence
to direct it to different sites in the DNA
where we, as scientists,
might want to introduce
a double-stranded break in DNA,
and actually have this protein go
to that site and create
the double-stranded break.
And so, just to show you
how this actually works,
and I'll say a little bit more about this
in a couple of minutes,
but this is 3D printed model
of the Cas9 protein in white,
with its guide RNA in orange
and a DNA molecule running
through the protein.
You can actually see this
DNA double helix coming in
as it enters the protein,
it actually opens up
and allows interaction of
this 20-nucleotide stretch
of the DNA with the RNA.
So you get a RNA-DNA
hybrid inside the protein.
The other strand of
the DNA is peeled away,
and this allows the two
molecular blades in the protein
to actually make a very
precise cut in the DNA.
Now, so this was sort
of the moment for us,
when this project went from being
a kind of cool, fun project to realizing
that we might be sitting on
a real exciting technology.
And the reason to think
that was that in parallel
with the very sleepy field of
CRISPR biology at that time,
was a lot of research that
had gone on over the past
couple of decades showing that
in animal and plant cells,
there's very sophisticated machinery
for detecting double-stranded DNA breaks
in those cells and fixing them.
And so when cells, if
this is genomic piece
of segment out of the genome,
genomic DNA,
if that DNA is broken at a site,
we have a double-stranded
break in the DNA,
these cells can repair that break
through a process called
nonhomologous end joining
that leads to a typically
a little disruption
in the DNA sequences.
The ends are pasted back together,
or if there's a donor
DNA template in the cell,
that molecule can be
recombined into the DNA,
if there's a little bit of
overlapping homologous sequence,
to actually introduce
new genetic information
at exactly the site
where the double-stranded break occurred.
And so, scientists have appreciated this
and had been working
away for awhile to try
to figure out how do we
introduce double-stranded breaks
exactly where we want to
make a change in the genome.
And so, some technologies
for doing this had come along
that were actually very promising,
and these are shown here,
zinc-finger nucleases, or talen proteins,
or homing endonucleases
are all protein-based
technologies that require
engineering a new protein
that can recognize a
particular DNA sequence,
and by coupling it to a
cleaving domain of a protein,
then you can have an engineered enzyme
that will actually make
a double-stranded break
in DNA exactly where you want.
And these can work very
well, and you might have seen
very recently in the news
a story about a child that,
a one-year-old child that had leukemia,
they were actually able to
use a talen-based strategy
to create a cancer immunotherapy
that was effective in her case.
So these therapies can,
these kinds of technologies
can be extremely powerful.
The challenge was that they're
also very labor intensive,
a new protein had to be
engineered for every experiment.
And so, it just meant that most
scientists around the world
had not been able to
adopt this technology.
It was too expensive, it
was too time consuming,
and it required too much experitse.
We thought, wouldn't it be cool
if you could use one protein,
never have to change it,
and redirect it to create a
targeted double-stranded break
by simply changing the
sequence of a short guide RNA
that is quite easy to do
by standard molecular biology methods?
And so that was exactly what we proposed
when we published this
work in the summer of 2012,
was that this could be
a very interesting way
to do RNA-programmed genome editing.
And so what happened next
was really quite amazing,
and that is that a number
of labs around the world
saw this paper and started to
test it in different systems.
And this is a slide I
got from my colleague
at North Carolina State
University Rodolphe Barrangou,
and showing just the sort
of exponential adoption
of the CRISPR technology
over the last three years
as people start using it worldwide
for different kinds of applications.
And in fact, even before the end of 2012,
there were six papers
submitted for publication
from different labs, including our own,
showing that you could use this technology
for engineering human cells,
but also for actually
engineering entire organisms
like zebra fish.
So it's one of the things
that's made it so exciting
is that it's a platform for
doing genome engineering
that works essentially in any
system where people tested it.
So, you know, people have asked me
why did this technology take off in a way
that these earlier technologies didn't?
And I think there are really
three answers to that question.
One is what I like to call
software versus hardware
where the older technologies
were protein-based
and effectively hardwired.
You had to recreate or
create a new protein
or pair of proteins for every experiment.
Here, we have something that is sort of
more analagous to software.
We can reprogram a single protein easily
using a small fragment of RNA.
So it just means that it's a technology
that is easily adopted and
tested in different systems,
and because it generally is
effective in these cells,
people have been able to employ it
for all sorts of applictions.
It works in, as I mentioned,
in essentially any animal
and plant system that's
been tested so far.
And as I want to share with you now,
that we know that this
has evolved in bacteria
for rapid and accurate DNA recognition,
which means that it's really very useful
for lots of applications
where you need precision
in the editing that you want to do.
So, I'm going to show you, I
want to show you a short movie.
If you can play the movie.
So this is sort of how we imagined
this might operate in a eukaryotic cell.
So here we are zooming into the cell,
and as we look inside the nucleus,
of course, in eukaryotic cells,
the DNA is highly packaged in chromatin.
So somehow this bacterial
enzyme has to deal
with DNA that's wound around histones here
to form these nucleosome structures.
It has to find a single target site,
for example, in a genome,
that has a 20 base pair match
to the guide RNA and the protein.
What it does, it opens up the
DNA and the molecular blades
in the protein make this
double-stranded break.
Now we have a break in the
DNA that has to be repaired
and the cell has machinery for doing that.
In this example here by
introducing or recombining in
a segment of DNA that
introduces new information
into this particular genome.
And it does that in a targeted fashion.
And so, you know, people
are now using this
to engineer plants,
animals, lots of organisms
that in the past were
genetically intractable
by using this kind of a strategy.
So, how does it work?
And that's something that my
lab is very keen to understand.
We feel like, you know, the
opportunity to understand
this at a molecular level
is not only interesting,
and reveals fundamental biology,
but it actually will be
very important for ensuring
that this technology is
really going to be useful
for doing things like curing
human genetic disease.
So one of the things that's
been very interesting
about this protein Cas9 is
that it's easily modified.
And you'll hear about this
later from Jonathan Weissman,
whose lab has done a lot of this work.
But basically it's been
possible to make this protein
into a version that is
deactivated for DNA cleavage,
it can bind DNA in a targeted way,
but not actually generate
double-stranded break,
and then that protein can also be coupled
to functional domains that
allow genes to be turned on
or turned off in a very specific fashion.
So that's a very powerful
application of this,
that doesn't involve any
genome engineering per se,
it doesn't involve making
double-stranded breaks
in the DNA of a genome.
And people have also been able
to use that kind of approach
for imaging, looking at
particular sites in a genome
by lighting them up using
a fluorescently-labeled
version of this protein.
It's naturally multiplexed.
And what I mean by that
is that in bacteria,
bacteria naturally will
program this protein
with a variety of different guide RNAs,
because they want to protect the cell
from multiple viruses at the same time.
That means as scientists
we can do the same thing as a technology.
We can program this protein
with multiple different guide RNAs
in the same experiment in the same cell,
and have multiple places in the genome
where we introduce precise changes.
So it's a very powerful way
to do a lot of experimentation
at the genetic level
in a single experiment.
And as I mentioned, it's evolved
for rapid and accurate
DNA target recognition.
And I would just like to share
with you a few recent sets
of data that we've obtained in the lab
that help us understand
how this actually works.
So one of the things
that emerged early on,
so this is a cartoon
that illustrates work that
we did collaboratively
with Eva Nogales, a professor
here at UC Berkeley,
who is a specialist in
cryo-electron microscopy.
We worked with her laboratory,
two graduate students,
David Taylor and Sam Sternberg,
who teamed up to look at
the Cas9 protein structure
as it goes from protein alone
to assembling with nucleic acids.
And what those experiments revealed,
even at about 30 angstrom resolution,
and not very high resolution,
we could see that this protein,
when it's in the protein alone state,
it starts off in a closed confirmation
in which the two sort of structural parts
of the protein are close together,
as soon as it assembles with guide RNA,
those protein lobes rearrange
to open up a channel
in the center of the protein,
which is where the DNA ends up
once this complex
assembles with a substrate
or a target DNA molecule.
So we already got the
sense even at this sort of
low resolution experiment,
that there was something
very interesting going on
with the way this protein rearranged
as it assembled with nucleic acid.
And now I want to show you a movie that,
hold on just a second,
I want to show you a movie
that is going to illustrate
the confirmational
changes now using a series
of crystallographic
structures available for Cas9,
so a much higher resolution snapshots
of this protein in different states.
And if we morph these together,
you can actually see
this protein undergoes
a remarkable structural change
as it assembles with nucleic acid.
So please, start the movie.
So this starts off with a
protein in the closed state.
As it morphs to the
structure bound to RNA,
you saw a big rearrangement in
this part of the protein here
to accommodate the RNA, and
here's the central channel
where the guide strand of the RNA ends up,
and that's actually where
the RNA-DNA hybrid will form
once the substrate binds.
Now when the DNA substrate binds,
there's an additional
confirmational change in the protein
to accommodate that RNA-DNA hybrid here
in the center of the protein.
And importantly, this
domain right here in yellow
is one of the catalytic cleavers,
this is one of the domains
that actually cuts the DNA.
And we found that in all
of the available crystal structures,
this domain is in the wrong place,
it's over here, it's in the wrong place
to actually cut the DNA.
It's about 30 angstroms away
from where it needs to be.
And so what you just saw was our imagined
sort of modeling of how this
domain would have to swing
into place to actually
be a functional cleaver
and to be able to cut the DNA.
And so what's emerged in
our recent experiments
in a paper that was just very
recently published last week,
we were able to show using
fluorescent dyes on this protein
that we can actually detect this series
of confirmational changes
by following changes
in fluorescence as these dyes
move into different positions
during this series of
confirmational changes.
And what we learned from that is
that we eventually do detect,
of course, this active state
of the protein, and we
think that that active state
only occurs when this protein is docked
on a DNA molecule that
has a perfect match,
or at least a very close to
perfect match to the guide RNA.
So it really has a way of
ensuring that it only cuts DNA
that is matched to the guide RNA,
otherwise it sometimes can bind to DNAs
that are off targets, that
don't have a perfect match
to the guide, but because the cleaver
is not in the right place,
the DNA does not get cut.
Okay.
All right, so the other
thing that we're doing,
so, you know, these experiments
that I've showed you
are all in vitro, that means we're working
with purified proteins, nucleic acids,
and doing experiments to try
to figure out mechanisms.
Of course, we would
really like to figure out
how does this actually
operate inside of a cell.
And if you think about it,
it's a really interesting challenge,
because this is a protein
that has evolved over time
in bacteria and so it has to
deal with bacterial genomes
which are a lot smaller
than eukaryotic genomes,
like the human genome, and
also don't have the kind
of highly compacted structures
that we see in chromatin
in eukaryotic cells.
And so how does a bacterial
enzyme deal with that?
And so this is just a picture
of a nucleosome
in the eukaryotic nucleus,
and here's the cartoon of the Cas9 protein
and what it actually has to do on the DNA.
So how does it do this when
it's got to deal with DNA
that's wrapped up like this?
And so we're sort of,
the questions we're sort of thinking about
is how does it deal not only
with chromatin structure,
but also the larger size
of mammalian genomes
as well as the sort of
overall organization
of DNA in the nucleus?
And so to start to answer those questions,
Spencer Knight, a graduate student here
who's jointly in my lab and
the lab of Robert Tjian,
has been working on a system
to detect and visualize
Cas9 complexes as they
search through the DNA
of a eukaryotic cell of a mammalian cell.
So I just wanted to show you
a little bit of his data.
So basically, what we can do
is we can fluorescently label
Cas9 proteins that are
inside living cells.
So we're looking, we're
visualizing live cell nuclei,
and we're watching particles
of the Cas9 protein
moving around the nucleus.
And when we program Cas9
with what Spencer calls
a nonsense guide, a guide
RNA that shouldn't recognize
any particular sequence in that genome,
we see very rapid movement
of those particles,
and we can plot this,
and we have the log of the
diffusion coefficient here
and the number of particles over here,
and you can see that most of
these particles are moving
very, very rapidly around
the cell with respect,
and certainly a lot faster than a protein
that is a part of the
chromatin structure called H2B
that has much, much slower
kinetics in the cell.
But we see a very interesting change
when we program Cas9 with a guide RNA
that recognizes about
300,000 sites in the nucleus.
So this is a highly repetitive element
in the mammalian genome.
And now we find that these,
a lot of these particles
are moving much more slowly
in the cell, you can really
see that visually here.
And if we plot these, we
see almost a, sort of a
two different populations of molecules.
Some that are still moving
with very fast kinetics,
but a large number now that
are moving very, very slowly,
and we think that these
correspond to particles
that have actually parked themselves
on a complementary sequence in the genome
and they hold on, they don't let go.
Actually, in these experiments we're using
a catalytically inactive
form of the protein,
so we're not actually cutting the DNA.
And so through doing
this kind of experiment,
we've been able to do things like measure
the kinetics of target search in the cell,
and we're working right
now to try to measure
how long it actually
takes a single particle
of Cas9 to find a single
target site in the human genome
and really understand how many molecules
do we really have to have in the genome
to get efficient editing.
And this should help us to
avoid off target effects.
So, finally, I just
want to sort of mention
what I think, sort of going forward,
what some of the
challenges are going to be
to using this technology.
And I think one of the big
ones is how do we deliver this.
The other big one I think
is how we control the way
that the DNA is repaired in
cells after the cut is made.
But certainly we have
to be able to deliver it
in the first place, get it into tissues,
and wouldn't it be lovely if
we could get it into tissues
in a specific fashion
so that only the tissue
where you want therapeutic
editing to occur will happen.
And so we've been thinking
about doing this, again,
from the perspective of biochemists,
we like the idea of using
a preassembled protein RNA
complex for delivering.
We call these RNPs that stands for RNA,
ribonucleic protein or
RNA protein complex,
and the idea is simply to
take purified Cas9 protein,
assemble it with one or more guide RNAs
that will direct it to sites
where we want to induce editing,
and then deliver it into cells,
and there are different ways to do that,
we're currently doing it
with a chemical strategy,
and we find that when
we do this with cells
cultured in a lab, we can
actually detect editing
within a few hours,
just a couple of hours,
sort of amazing to think about
that in this protein is able to search
through the human genome, make a cut,
and the cut will be repaired
in a way that we can detect
within just a couple of hours.
Secondly, we know that the half-life
of the ribonucleic protein
complex is about 24 hours.
And that, we think, will really
minimize off target effects.
And finally, and this is the work
of both Brett Staahl and
Steven Lin in the laboratory
who have really been
developing this methodology.
The idea was to take
this approach and use it
to co-deliver DNA templates for repair.
And the idea there is to help
control the repair pathway
by having the template
for repair be present
with the tool that's doing the cutting.
And Steve and Brett have
seen really nice results
using this kind of strategy.
And we've also been working closely
with Alex Marson and Jennifer Puck,
who are immunologists at UC San Francisco,
to use this for editing
primary human T cells.
And in partnership with their labs,
we've been able to show
that we can actually edit
human T cells, not only to
make knock outs of genes,
but actually to make knock ins
using this kind of strategy.
So, there's a lot of
excitement about this,
because, of course, this opens the door
to being able to edit these immune cells
in a targeted and precise
fashion for doing things
like cancer immunotherapy and, of course,
for lots of research purposes as well.
And Jacob Corn, I want to mention as well,
has been a part of this project,
as part of the Innovative
Genomics Initiative
that we have going.
It's a partnership
between Berkeley and UCSF.
So, I just want to close by
sort of closing this question,
What should we do now
that genomes can be
edited relatively easily?
And I started really
thinking about this a lot
in the first few months
after this technology was out
and being utilized by more
and more laboratories,
and realizing that there was
the potential to do a lot
of very exciting things with
this, but also some things
that maybe we should be thinking about
in a more cautionary fashion.
And I realized that the science was moving
at a thousand miles an hour
with papers coming out, you know, daily,
and accelerating, as I showed you,
and meanwhile, most
non-scientists were really unaware
of what we were all doing in our labs.
And so I decided to really get out
in front of that conversation.
And so with the Innovative
Genomics Initiative,
and Jonathan Weissman
who's a part of it here,
we decided to convene a meeting
in the early part of 2015
with a few scientists
from around California
and a few from elsewhere in Boston,
to come out and discuss this question.
And the upshot of that
meeting was to publish a
what we called a prudent path forward
for genome engineering
that particularly focused
on editing the human germline.
That means making changes, precise changes
to human embryos, or eggs or sperm,
in such a way that those
changes could be passed on
to future generations.
And this has led to a call
for a global conversation
about this in the scientific
and broader communities,
and next month in December there will be
the first International
Summit in Washington
sponsored by the Chinese
Academy of Sciences,
the National Academy of
Sciences in the U.S.,
and the Royal Society in the U.K.
with lots of participation from
scientists around the world
and others who are interested to come
and discuss this very issue
and how we should proceed
in a safe and appropriate fashion.
So stay tuned for that.
And I'd like to close by thanking my lab.
This is really a great group of people.
I tried to mention all of
those that were involved
in this research along the way.
I see some of them in the
audience, that's great.
And, of course, I want
to give a real shout out
to our wonderful collaborators,
including the folks at UCSF,
this has really been a great opportunity
for a basic researcher like
me to take a technology
that grew out of our sort
of home grown lab efforts,
and see it really evolving into something
that I think is going to be
really impactful in the clinic.
And then of course, we couldn't do any
of this without funding.
We're extremely grateful
to all of these groups,
and in particular to the
National Science Foundation
who gave me a very small
grant to support one student
that allowed us to get
going on this project
before anybody knew it would turn
into this kind of technology.
So, thank you very much.
(audience applauds)
- [Voiceover] We have some
time for some questions.
- I see one in the back over there.
- [Voiceover] Hello, hi.
With the last paper that you referenced
about the social implications
of this technology,
are you kind of alluding to the idea
of editing genomes of human embryos
or existing cells that would,
like in that are in storage right now,
what exactly are the consequences of this?
- Right, well, so I
think just to be clear,
there's, you know, sort of important
to make the distinction
between editing cells
in an adult, right, somatic cell editing,
which means that we could make changes
in, say a tissue, that would
have a therapeutic benefit,
but not in a way that those
changes would be passed
on to children, right.
Whereas germline editing
refers to making changes
such that the eggs and
sperm of that person,
that would develop from that edited embryo
would contain those changes.
So they would be passed
on to future generations.
And we're really proposing
that that latter type
of editing is something
that really raises ethical
and societal questions
and needs to be thoroughly
considered before proceeding.
- Jennifer, maybe I can ask the,
one of the big challenges is to make sure
that you don't have off target effects,
does the structural
analysis explain to you
how even a single base mismatch
could prevent the cleaving,
for example, or prevent the function?
- Yeah, I think one thing
that's very interesting is
that the series of confirmational changes
that happen in the protein
are really happening
as a function of cognate base pairing
between the target DNA and the guide RNA
such that we even see that when there's a,
when there's a mismatch
in the RNA-DNA hybrid
that occurs at a certain
one end of that duplex
that's a little more tolerant
to mismatches than the other.
But even at that more tolerant end,
we see that there's an effect
on the ability of that
confirmational change
to put the cleavage domain
in the right place to cut the DNA.
And we know this from our
fluorescence-based studies.
So I think the answer is,
yes, and I'm really interested
to see if we can now take
that mechanistic understanding
and use it to help either
design or evolve protein
that maybe have even better accuracy.
- I see a person over here.
Over there.
- A couple, yeah.
I don't have much time.
- [Voiceover] Anyway, I'll talk loudly.
Your picture looked like
that the Cas9 was popping
on and off the DNA
rather than sliding along it.
Was that just because there was
such a small plane of focus,
- No.
- [Voiceover] Or do you
think it pops on and off?
- That's what we think.
All of our, both our in vitro and
our cell-based data are consistent
with the diffusion models.
So it's basically diffusing
rapidly around the cell,
binding and releasing
the DNA very quickly,
rather than sliding
processively along the DNA.
- I think that maybe one more question.
- [Voiceover] Yeah, I
had a question related
to the off targets, so you mentioned
that some of the specificity comes
from translocation of the cleavage domain,
and so do you think that
all of the technologies
that use Cas9, or like a
Cas9 that doesn't cleave,
might actually have difference of targets
than sort of like traditional
double-stranded break?
- Right now I would
answer it, I would say no.
And the reason is that these
deactivated forms of Cas9
that people are working with
are point mutations in the active site.
Okay, so like a single amino acid change,
so that's a big domain that
has to swing into place,
and we think,
we've not really tested these,
let's see, I'm trying to think
if we have done the FRET.
I think the FRET experiments
have not been done
with the deactivated form of
the protein for the most part,
but we have no reason to
think that that domain motion
in the protein wouldn't happen,
even when you have a point mutation
in the DNA or in the protein.
But that might be something
that would be interesting
to look at actually, yeah.
- Thank you.
(audience applauds)
