(light futuristic music)
- So Dana's given a great introduction
to gene editing
and talked about some
of the nuts and bolts
of how it actually works in cells
and what I thought I would do
for the next 40 minutes or so
is tell you about the background
to the CRISPR technology.
I think there's a couple of
really interesting things
to appreciate about this.
First of all, the word
CRISPR has become a verb.
You hear people saying to CRISPR
and I wanted to sort of
tell you a little bit about
where this all came from
and I think it illustrates the way
that new technologies often emerge.
They come from unexpected directions
by people that are not necessarily trying
to discover new technology.
They're working on fundamental questions
and they come across, in this case,
a mechanism that reveals itself
to be something that can be harnessed
for a very different purpose.
So I'm gonna talk about that
and then I'm gonna say a little bit
about where I think this is all going.
So the CRISPR field
is a very young field.
It's only really been
around since the mid-2000s
when scientists discovered
that a lot of bacteria have
repetitive DNA sequences
in their genomes
that came to be called CRISPRs,
an acronym that stands
for clusters of regularly interspaced
short palindromic repeats,
a big mouthful.
What does that mean?
Well it just means that many bacteria
have a distinctive
feature in the chromosome,
sometimes more than one,
that include repetitive sequences
shown by the black
diamonds in this cartoon
that flank unique sequences
shown by the colored boxes
and three research teams in the mid-2000s
noticed that in many cases
these unique sequences
in the CRISPR arrays
come from DNA found in viruses.
So this was the first indication
that these might in fact be some kind
of acquired immune system in bacteria
and scientists also noticed
that together with these arrays
were CRISPR-associated or cas genes
typically located nearby
in the bacterial chromosome
that turned out to be encoding proteins
that are part of this
adaptive immune system.
And our own research at Berkeley
started because of the
work of Jillian Banfield,
the scientist here at
Berkeley who does research
on bacteria, typically on
uncultured, unidentified bacteria,
and her research uncovered the fact
that many bacteria that you can isolate
in various environmental niches
are very abundant in these elements
and so they are probably
very actively using
this kind of immune system
to protect themselves
from viruses in the wild.
So it's some very interesting biology
and so I'm gonna show
you a very short video
that illustrates how
these actually function
and how the activities native
to these bacterial immune systems
are very very nicely relevant
to the work that Dana just talked about
because what they do
is they make targeted cuts in DNA.
So in bacteria this is happening
to protect cells from viruses but we can,
as you heard very nicely from Dana,
this can be harnessed in
various kinds of cells
per gene editing.
So I think my,
let's see if the audio works here.
It's not, okay.
Well we saw, I'll narrate this.
So we have viruses that
are entering a cell.
You can see a virus injecting
its DNA into a bacterium
and if this bacterium has a
CRISPR array in the genome
it can acquire new pieces
of DNA from the virus
and integrate them into the array,
keeping this pattern of repeats.
And the cell is then able to
make a copy of that sequence
in the form of an RNA molecule
that gets subsequently broken
into smaller units that each
include one of the sequences
derived from a virus
together with the sequence
coming from the repeat.
And those RNAs are then kind of marked
as CRISPRs molecules by
the presence of this repeat
and they combine with a
second RNA called tracer
to form a structure that binds
to the protein called Cas9.
So this RNA protein complex
then is able to survey the cell
looking for DNA sequences
that have a sequence matching
the sequence of the RNA
and if that match is found
then this protein RNA complex
unwinds the DNA locally
to allow RNA/DNA hybridization to occur.
A double-stranded break
is induced in the DNA
and if this happens in a bacterium
that leads to degradation
of the viral DNA.
But if you introduce that
system into a eucaryotic cell,
a plant or animal cell
as you heard from Dana,
these cells have sophisticated machinery
to repair double-stranded breaks
and they can do so by
introducing a small change
at the site of the break
or even by integrating a new piece of DNA
during the process of repair
and so that means that this method
of introducing double-stranded
breaks in a targeted fashion
into the genomes of plants and animals
is a very effective way to
conduct genome engineering.
So that's a very fast summary
of why this system was
harnessed as a technology
and why it's been so useful,
why it's really taken off
over the last five years
and has spread through many
different areas of biology
enabling all sorts of
research and now applications
that would have been very difficult
if not impossible to do in the past.
So I'm gonna just I really
wanna tell you three things.
I want to talk a little
bit about the mechanism
of this process,
how this RNA-guided DNA
cutting actually works.
Why do we care?
Well, I'm a biochemist
so I think it's just kind of cool biology
but as you'll see I think once
we understand how it works
we can actually harness that activity
and we can think about
ways to make it better
for applications that
we want to use it for.
So I'm gonna tell you a
little bit about this.
I want to tell you a
little bit of new biology
so one of the things that's
happening in the field right now
is continuing investigations
not only of new CRISPR pathways
but also of how these are interfacing
with other proteins that
are found in bacteria
and one of the very recent discoveries
has been finding that many cells
and the phage that infect them,
the viruses that infect them,
have what are called anti-CRISPR proteins,
these are proteins
that actually inhibit the CRISPR pathways,
and these are turning out also
to be potentially very
useful as technologies
and I'll give you one
brief example of that.
And then at the end I
wanna just say a little bit
about what we think about
the societal implications
of a technology that is so enabling
for making targeted changes
to the DNA of cells and organisms.
It's really a profound
thing if you think about it
'cause it really gives
humans now the power
to control evolution of
organisms in our environment
and also potentially our own evolution
in a very targeted fashion.
So it's exciting, it's enabling,
it's moving fields
forward incredibly quickly
but it also brings sort
of this sense of awe
and the feeling that we need to proceed
with appropriate caution and respect
for this powerful technology.
So I'm gonna say just a few words
about that towards the end.
Okay but let's talk first about CRISPRs
and how they operate.
So this is a slide that shows the pathway
of adaptive immunity in cells.
So one of the things
that was very exciting
in the very early days of the CRISPR field
was the idea that bacteria
actually have an adaptive immune system,
kind of analogous to the way that we have
antibodies, we have, of course,
very sophisticated adaptive immune system
that allows us to protect
ourselves from pathogens.
Nobody thought that bacteria,
very simple, single-celled organisms,
would be capable of
that kind of adaptation
but it turns out that they are.
However, that pathway
works very differently
from the way that our own
adaptive immune system works
and it works by means of
these acquired sequences
that are integrated into the CRISPR arrays
as I showed you in that video
and then these become
templates for RNA molecules
that are transcribed
that include copies of all
of these integrated bits
of viral DNA together with the
associated repeat sequence.
And note that as the
CRISPR acronym indicates
these repeats are often
palindromic in character.
That means that when they're made
in a single-stranded form like
in the RNA molecule shown here
they can fold back and form structures
like little hairpins
that are recognized by some
of the CRISPR cas proteins
and that's actually how these
RNAs are often processed
is by the recognition of those sequences.
So the cell very nicely
effectively labels these
as CRISPR RNAs
by using these unique
sequence and structure tags.
And after the RNA molecules are processed
into individual units
that each include a
virally-derived RNA sequence
they combine with one or more cas proteins
to form surveillance complexes.
They can search the cell
looking for matching DNA
or sometimes RNA sequences
leading to degradation by cutting
of those matching nucleic acids.
So it's really a process of adaptation,
acquiring these sequences from viruses
that are entering the
cell for the first time,
expressing those sequences
in the form of RNA
and then using the resulting
protein RNA complexes
to interfere with foreign nucleic acids.
And so I'm gonna say, I'm gonna
tell you a little bit more
about this step here, interference,
because this is really the step
that through understanding how it worked
allowed this system to be harnessed
as a gene editing technology
as you heard about from Dana.
Now one of the things to
appreciate about CRISPRs
is that there are many
flavors of these pathways
and this is a cartoon,
it's actually already out of date
but it was taken it from a
review published in early 2016
that shows different examples
of CRISPR systems
that are classified according
to the numbers and types
of cas proteins
that are found in these systems
and the main thing I want you to note here
is that we can really
classify these systems
in two broad categories
called Class 1 and Class 2.
The Class 1 systems all
include multiple genes,
multiple cas proteins,
that have to be present in the cell
and have to assemble with CRISPR RNAs
to form surveillance complexes.
In contrast, the Class 2
systems of CRISPR systems
include a single gene, typically,
that encodes one large protein
that is able to combine with CRISPR RNAs
and provide the cell with protection.
And it was really through
studying these systems
that it was possible to
harness these proteins
for gene editing purposes.
And this is a slide that,
this is just showing,
this was from a recent
conference that we attended
that really just shows
examples in cartoon form
of these different classes of systems.
Class 1 over here including
large arrays of proteins
that assemble with CRISPR molecules
to form these surveillance complexes
and then over here single proteins
that combine with RNA to carry
out that kind of function.
And so in the early days when research
had focused initially
on these Class 1 systems
they were very interesting,
the functions were fascinating
but it was hard to imagine how
you could really harness them
as a technology
because you would
require multiple proteins
to be made in cells
and to assemble and it just seemed
like a many component system
that might be very
complicated to get working
in a heterologous cell type
whereas work on these systems over here
which are much simpler
and were simplified further
by understanding that you could combine
the two natural RNAs called
CRISPR RNA and Tracer RNA
into a single guide form,
this converted what had
been initially a very,
looked like a pretty
complicated kind of system
into something that looked a lot easier
to harness as a tool.
And so for us
one of the key questions
that we set out to address
in those early days
was to understand the
function of the first example
of this large type of
protein, single protein,
in the Class 2 CRISPR systems
that could operate to protect cells
in an RNA-guided fashion,
protect them from viral infection,
and the question was
really what is the function
of this encoded protein
which in the very early
days was known as CSN1
and then very quickly became called Cas9.
And that line of research
which was conducted with a collaborator
Emmanuelle Charpentier
and her student Krzysztof Chylinski
revealed that Cas9 is an amazing enzyme
that has the ability to
recognize double-stranded DNA
at positions in the DNA sequence
that match a 20 nucleotide sequence
in this guiding RNA
and note that I'm showing you here
the guide RNA in its single-guide form
where the natural CRISPR RNA
which would be this part
of the sequence here
and what's called the Tracer RNA
which would be this part
of the sequence over here,
have been combined by linking
them together covalently.
So this can be made as
a single transcript,
something that turned
out to be very helpful
in harnessing this as a technology
because it's made it easy
for researchers to make RNAs
that would have a desired sequence here
that would track Cas9 to a
particular site in a genome
and trigger its DNA cutting ability
allowing it to make a
blunt double-stranded break
at a precise place in a DNA molecule
or in an entire genome
and you've already heard quite
a lot about that from Dana.
And so in our lab we've
been sort of curious
about really addressing this question
of how recognition really works,
not just that we can see
that involves base pairing
but it's really also a question
of how this protein is
able to open up the DNA
in a genome
and you've gotta imagine
that you've got the DNA in
a typical eucaryotic cell,
of course it's in the nucleus,
it's also highly packaged.
It's wrapped around histones,
it's compacted into chromatin,
different types of chromatin,
lots of proteins around
are bound to it,
DNA replication and repair machinery
and other proteins moving through.
How does this bacterial protein
deal with all of that
and somehow find these sequences
typically quite accurately
and generate double-stranded breaks?
And that's really the question
that we've been seeking
to understand over the last few years.
And I threw this slide in
to remind me to tell you
that unlike this sort
of cartoon right here
the Cas9 protein has to
be able to unwind DNA
without any external source of energy.
So it doesn't hydrolyze
ATP or GTP to unwind DNA.
Somehow it triggers DNA unwinding
by some other mechanism.
And so we'd like to
understand how that works
and why do we want to know this?
Well again, we're very
interested to understand
what makes this enzyme
functional in eucaryotic cells,
how does it deal with
chromatin, et cetera,
how does it get to the
right site in the cell,
what happens when it
gets to the wrong site?
Can we do things to
prevent it from accessing
the wrong site, make it even more accurate
than it is naturally?
And also can we harness its
DNA recognition activities
to do other things
like not just trigger covalent changes
to the DNA in the genome
but also to recruit other factors
to positions in a genome,
use it for DNA imaging, for example,
and also for the kind of thing
that Luke Gilbert is doing
which is harnessing its
activity to regulate
the expression of genes,
so making changes to the
levels of proteins in cells
without actually changing
the DNA sequence in cells.
So one of the things that's
emerged over the last few years
is that one of the ways
that Cas9 probably does this
is through its ability
to change confirmation
upon binding to nucleic acid
and so I'm gonna show you a little video
that was made by a student Ben LaFrance
that morphs together a series
of crystallographic structures
solved for Cas9 in
different states of assembly
with nucleic acid.
And so this is a movie that
starts with Cas9 protein alone,
that's a crystallographic structure,
and you see it morphing to
the state of the protein
when its bound to the orange guide RNA
and you saw a big rotation in
this gray part of the protein
that reorients it to open
up a channel in the center
where the guide part of the RNA is sitting
and then when this protein
binds to a DNA molecule
you can see additional structural changes
that happen in the protein here
to accommodate the
RNA/DNA hybrid that forms.
Now those structures showed us
that there had to be an
additional structural change.
This rotation that
you're seeing right here
which was modeled in at the
time that we made this video
because we knew that
this part of the enzyme
which is a domain in
the protein called HNH,
this is actually one of the
chemical cleavers in Cas9.
It's the part of the protein
that cuts the DNA strand
that base pairs with the guide RNA.
But in all of the crystal structures
that were solved initially,
which were solved using
single strands of DNA
that were annealed to the guide RNA,
this domain was not in the right place
to actually cut the DNA.
It was located quite far away.
It was sort of over here
rather than in this position
you can see modeled here
where it would have to be positioned
to actually make a cut in
the targeted DNA strand,
so what was going on,
and so one of the things
that emerged more recently
in research that was done
by a team at Berkeley
including Fugo Jiang and
David Taylor in my lab
and this was a very nice collaboration
with the lab of Eva
Nogales here at Berekeley
who does cryoelectronmicrosopy,
was at a combination of crystallography
and electron microscopy
allowed these students to trap a structure
of the Cas9 protein bound
to its true substrate
which is a double-stranded DNA molecule
and so you can see
that hopefully it's a little bit dark here
but hopefully you can see
this double-stranded DNA.
So here's the one strand is in blue
and the other strand is in magenta
so you can see the duplex DNA here
opening up inside the protein.
This targeted strand is forming a duplex
with the guide RNA
and then here's the non, what
we call the non-target strand
is over here
and remember that when Dana
Carroll talked about work
that Chris Richardson is doing
at the IGI with Jacob Corn
they had found experimentally
that this part of the DNA
is more exposed after DNA cleavage
and this structure kind of
reveals the basis for that.
You can see its located really
on the surface of the protein
rather than buried inside
like this strand over here.
And the cool thing about this structure
was that we found that the,
and I think this is actually an animation,
I could show this to you,
but it's basically,
I think I'll just tell you,
that this is a, if you
look at this green part
of the enzyme here,
in this structure this domain
that cuts the target DNA strand
is now actually positioned
very near where it needs to be
to cut the DNA.
So there's something about having
the non-target strand
present in the complex
that actually triggers this protein
to be in the right confirmation
to actually conduct chemistry.
And so we're, we're
doing a lot of work now,
and I'll tell you a little
bit about this shortly,
to really understand
what triggers this domain
to swing into place to cut the DNA
because there's a lot of evidence
that this is a protein that
can bind DNA quite readily
even in cells
and people have done experiments showing
that you can detect binding of Cas9
at a lot of sort of close
but not quite perfect matches
to the guide RNA and cells
and yet most of those sites
are never altered chemically.
Why is that?
Well we think one reason
is because this domain
is actually very sensitive
to full base pairing of the guide RNA
with the target DNA sequence.
And just to show you
why we think that's true
I'm gonna show you one
series of experiments
that are being done
to test the conformational states of Cas9.
So you might,
if you don't do biochemistry
or think about proteins
in this way
you might wonder how do we know that,
how can you actually figure
out structural changes
in proteins, I mean, that
sounds kind of detailed
and certainly we can't get
that information necessarily
from snapshot structures like
we see in crystallographic
structures of proteins.
And so one strategy
for testing conformational
changes in proteins
takes advantage of the
real physical changes
in distances between atoms,
or individual amino acids,
in a protein if there's a
change in its structure.
And so when we had this model
for what we thought must be
happening in the Cas9 protein
with respect to this active
site, this HNH domain
that had to swing into place
to cut the target DNA strand
the idea here was to introduce
pairs of chemical dyes
on the surface of the enzyme
that would be in very
different spatial relationships
depending on the
conformational state of Cas9.
So this is just showing an example
where we had this HNH domain in yellow
so the inactive state is shown over here,
we've got a dye sitting right there.
When this domain swings into
the active position shown here
you can see that the position of that dye
moves quite a lot
and if you've got another
dye sitting over here
in the protein
these two start of
initially very far apart,
they can't really chemically interact
so you don't get much of a signal
by something called
resonance energy transfer
but when this domain change happens
now these two dyes are very close together
and you can detect a
resonance energy transfer
between the two.
So that's one way that you can detect
these kinds of structural changes.
And just to show you a little bit of data.
This is actually fairly new,
I don't think this
publication has appeared yet,
and this is a collaboration
with Ahmet Yildiz,
a biophysicist here at Berkeley,
and his student Yavuz and a
student Janic Chen in my lab.
What these students did
was they were able to set up a system
where they can tether the
Cas9 guide RNA complex
to a surface
and they do this by, so
here's the guide RNA,
you can see it's RNA end
is being chemically linked
to this slide surface,
and then we can flow in
double-stranded DNA substrates
for a recognition by this complex
and we are using Cas9 proteins
containing these pairs of
dyes that I showed you.
So we can actually monitor changes
in fluorescence resonance
energy transfer called FRET
as a function of interactions
with these DNA substrates
and by setting up an experiment like this
it's very trivial to
change the DNA sequence.
So we can have DNA molecules
that have a perfect match
to the guide RNA
and then we can also test DNA molecules
that have various
mismatches to the guide RNA
and see how that affects the ability
of the protein to interact.
And I'll just show you
a little bit of data.
So what does the data for
these experiments look like?
So here we've got the Cas9 RNA/DNA complex
that we're monitoring formation of
and over here you can see
we're plotting the resonance
energy transfer signal
that we're getting
as a function of the complementarity
between the DNA molecule
we're using in the experiment
and the guide RNA.
So if you just look at this last line here
this is what we see if we use
just the Cas9 RNA complex,
no DNA is present.
The protein is in an inactive state
as I showed you before
and we get a FRET signal
so most of the particles here
have a very low FRET signal.
That's because the dyes are very far apart
in the confirmation, they don't interact.
As we add DNA molecules
that have increasing
amounts of complementarity
all the way up to a perfect
match, to the guide RNA,
you can see that more
and more of the molecules
are populating this active state
where the dyes now are very close together
because this conformational
change has happened
and so we're getting a very strong signal
between the dyes.
So we can really monitor that very nicely
and the cool thing in this experiment
was that we found that a lot
of the molecules initially
these proteins, get stuck in
an intermediate confirmation,
it's not fully off,
it's not fully inactive
but it's not in the active state either.
So there's sort of this intermediary state
that is populated and sort
of the degree of population
of this state depends,
again, on the complementarity
between the DNA and the guide RNA.
So it really tells us
that this is a protein
that's a sensor, right,
it's really sensing
the degree of a match
between a target DNA and the guide RNA
and that sensing is being conveyed
in terms of this conformational
change of the protein.
And so that really led to the idea
that the protein goes
through this series of steps
to get to an active state
in which it starts of
in this inactive form,
it goes through an intermediate
and then it reaches this
sort of fully-docked state
and then is able to cut the DNA
and this can then dissociate
and the protein gets reset
and can cut other DNA molecules
in a catalytic fashion.
And what we're doing right now
is we're actually using
this to design versions
of this protein
that are even more accurate,
they're even better
sensors of the target DNA
than occurs in nature
and we have a paper that we
just posted on the bio archive,
the pre-print server,
that shows design of new
mutations in the Cas9 enzyme
that we think are making this protein
an even better sensor,
potentially more accurate at DNA cutting
and thereby may be useful as a technology
for gene editing as well.
Okay, so let's talk a little bit
about how nature fights back.
And so the story of anti-CRISPRs
really started kind of where
the story of CRISPRs did
which was with microbiologists
who were trying to understand
how these systems operate in nature
and of course when
there's an ongoing battle
between infectious agents and their hosts
they're going to be protein,
there's a lot of selective pressure
for both systems to
evolve ways to get around
the defenses that are put up in each case.
This is a cartoon that was actually made
by Megan Hochstrasser
who is one of our outreach coordinators
here at the IGI
illustrating sort of in fanciful form
different ways that we could imagine
that cells might come up with ways
of blocking these CRISPR pathways.
They could have ways of
stopping the CRISPR proteins,
they could prevent binding
to the RNAs potentially
and there might even be other pathways
as I'll show you
that would lead to
inhibition of CRISPR systems.
And so Joe Bondy-Denomy,
a scientist at UC San
Francisco across the bay,
is one of the lead scientists
whose been working on
these anti-CRISPR systems.
So he's a microbiologist
studying ways in which phage interact
with their host bacteria
and he noticed that in,
there were sort of interesting examples
of organisms that should have been
or phage that should have been eliminated
by these CRISPR pathways
that weren't somehow.
And so why was that?
And investigating the mechanism
that led to the discovery
that in many cases these organisms,
these host organisms,
actually encode little proteins
that turn out to be inhibitory
to the CRISPR system.
And so as mechanistic biochemists
we've been very curious to understand
again, how do these work,
and how can these proteins
which turn out to all be very small,
they're typically under 100 amino acids,
how do these actually
operate as inhibitors
and can we use their mechanisms
to tell us more about the
way that these proteins,
in particular Cas9
enzymes, actually operate.
And I'm just gonna show
you a little bit of data.
Again this is unpublished, very new data.
But one of the things
that was very interesting,
and this work being done by students
Kevin Doxzen, whose here
and how working at the IGI,
and Lucas Harrington,
was they noticed that one
particular anti-CRISPR protein
that I'll abbreviate C1
turned out to have the
ability to block DNA cutting
and the way we can test this biochemically
is doing an experiment
where we can put a radioactive label
on one end of one strand
of a double-stranded DNA target sequence
and then we use this labeled DNA
in an in-vitro DNA cleavage experiment
and so if we do this with
a particular Cas9 enzyme
you can see we get very
nice cutting of the DNA
as you would expect if we don't
have any inhibitor around.
As soon as we add the inhibitor though
we see that DNA cleavage is blocked
and so once you see that
as a biochemist you say well,
that could mean a couple of things.
It could mean that this inhibitor
just now doesn't let the
Cas9 protein even bind
to the DNA substrate.
Maybe it just prevents binding
or it could be that it allows binding
but somehow prevents DNA cutting
and so to distinguish between
those two possibilities
Kevin and Lucas did an experiment
in which they used a
non-denaturing gel system.
So this is a gel system
that allows us to visualize
any trapping of proteins
as they associate with nucleic acids.
So if you look at the left
hand part of this gel system
here we're just doing,
we're taking these reactions right here,
no inhibitor is present,
and we just apply them to
this native gel system.
So we've got our DNAs radio labeled
and you can see that as we
increase the amount of Cas9
and its guide RNA that
we add to the reaction
over time we get cutting of the DNA
and the DNA dissociates
and you can see it running faster on this,
migrating faster on this gel system.
So that's just what you
would expect to see.
What happens when we add the inhibitor?
Very interesting result.
Now what we found was that
we saw not only no cutting of the DNA
but we saw that this radio labeled DNA
becomes trapped in a much
slow migrating product
and that turns out to
contain the Cas9 protein
and the guide RNA.
So that tells us that this inhibitor
doesn't prevent binding to the DNA
but somehow it prevents
the DNA from getting cut.
So it seemed very interesting.
How would that work?
And so to sort that out
Kevin and Lucas and
another student Josh Cofsky
made a whole series of
variants of the Cas9 protein
and I really just want
to point out one thing.
So these are just cartoons
that show kind of a linear cartoon
of the Cas9 protein sequence.
And what we found was that in every case
where the HNH domain was
included in the construct
we saw that there was binding
to this anti-CRISPR protein.
So this is just looking
at direct protein protein interactions
but constructs that were
missing the HNH domain
like this one right here
had no binding to the inhibitor.
So it really looked like
that was somehow the domain
responsible for this
protein protein association
and just to really bring that home
they made constructs
that are shown down here
that were cymeras
in which the HNH domain from
this inhibited Cas9 enzyme
was swapped into a different Cas9 protein
and this now becomes capable
of binding the inhibitor
whereas if we do the opposite
we use the Cas9 protein
that naturally can bind the inhibitor
but we replace the HNH with
a different Cas9's domain
that doesn't bind the inhibitor,
now it doesn't bind anymore.
So it really looks like it's specific
to this catalytic domain of the enzyme.
And so Lucas and Kevin were able to solve
a crystallographic structure
of that interaction
and this turned out to show
that the actual chemical,
chemically-important residues
in this HNH domain are physically blocked
by this little inhibitory protein.
It literally grabs on to the
sites important for chemistry
in this domain
and prevents them physically
from interacting with DNA
and the way we think this actually works,
and this is now a model,
is that this inhibitor grabs
on to the HNH domain in Cas9
and literally prevents it
from swinging into place
and cutting the DNA
as I showed you that we know
it has to be able to do.
So this is a really cool example
of a natural inhibitor
that has the ability to trap the protein
and its guide RNA on a DNA target
by not physically preventing
it from associating
but actually just taking advantage
of the natural mechanism of cutting
and preventing this conformational change.
So kind of cool mechanism
but again, do we care about this
from a technological standpoint?
And I would argue that we do
and it really comes back to the work
that Luke Gilbert and his
colleagues at UCSF have been doing
and also the work
originally of Stanley Chi
who is now at Stanford
who were able to show that
you could make forms of Cas9
that were inactivated
by making just targeted changes
to the chemically-important
residues in the enzyme
including in this HNH domain.
And then linking effector
proteins to the enzyme
that had the ability to repress
or activate transcription.
And so this shows that,
this is sort of how one can do this
kind of artificially in the laboratory,
but what if it's true that,
what if it turns out to be the case
that in phage or bacteria
that have this particular
anti-CRISPR protein
they can actually block
DNA cutting by Cas9
and thereby allow it to
function in a regulatory fashion
kind of analogous to this.
So that's something
that we're now testing.
So it may be that this
really kind of allows phage
or bacteria to expand the function
of Cas9 naturally in cells
not by making mutations in the enzyme
but simply by blocking
its ability to cut DNA
but retain its ability to
bind in an RNA guided fashion.
Okay, so just in the
last minute or two here
I just want to say a little bit
about what I call responsible progress
and so one of the things
that's been very exciting
over the last few years
is just the rapid adoption
of the Cas9 technology
for all sorts of gene
editing applications.
And this was a cartoon
that was published in Nature last year
just showing that as Dana said
now it's sort of over 100
organisms and cell types
and many many more cell types
that have been modified using this system.
And just to show you some
of the very recent things
that have been happening that
I think are really exciting.
So this was a paper that came out.
This was actually on the cover
of the journal Cell recently
where a group at Coldsfer and Harver
was able to use the Cas9 system
to make targeted changes in tomatoes
and they were doing this
to actually separate
two genetic changes in these plants
that had been selected by
traditional plant breeding
but couldn't be separated by
traditional plant breeding
because of the length
of time it would take
to actually do that.
They were able to knock
out one of those genes
and thereby create plants
that have much stronger branches.
They were able to hold
heavy fruit loads like this,
very practical use of gene editing
and doing something that
would have been difficult
if not impossible to do
with traditional methods.
Why was this on the cover of Cell?
Well I think it just
is sort of a harbinger
of where this is all going,
that in the future it's going
to be possible, we think,
to make these kinds of changes
in all sorts of plants,
not just the major crops
but also in plants that people are growing
in their gardens
and to do things that will create
traits that are very
useful in different ways.
Another recent example
was this one right here.
So this was a Chinese group
that published work
showing that you could actually cut out
integrated HIV proviruses
from the genomes of mice
and thereby effectively kind
of cure individual cells
of viral infections.
So it's kind of very reminiscent
of the way that this is
actually operating in nature
except here we're doing it in mice.
Now will this be an
actual therapy for HIV?
Probably not, right, because
it might be very hard
to actually cut all the proviruses
out of infected cells in these animals.
On the other hand,
it shows the precision of the technology
and it also points to the fact
that there's a lot of interest
in using this system
to protect organisms from viruses
by harnessing these activities
in ways that are analogous to the way
that they operate in nature.
And finally then there's germline editing
so all of the editing that's
being done at the moment
for clinical use
is being done in somatic cells
and cells that are fully differentiated
but we know that this
technology works very nicely
also in stem cells.
It works in germline cells.
This is an example from Russell
Vance's lab here at Berkeley
showing a fertilized mouse
egg held by a pipette
and we're seeing a needling coming in
injecting Cas9 guide RNA complexes
into this very early embryo
and when those edits are made to the DNA
they become heritable, right,
they become part of the entire animal
including its germ cells
so they can be passed on
to future generations.
And if that's done in a human being
then we have a situation
where we're making changes
that become embedded
in human populations in principle.
And so this has triggered
a lot of discussion
around the world,
very active conversations
about the ethics of this
and has led to the release very recently
of this document here by the
National Academy of Sciences
on human genome editing
which if anyone's interested
you can download it
and it really goes through all
of the implications of this,
especially for human germline editing,
and how sort of a roadmap
for how we as a scientific
community, global community,
can move ahead to ensure
that there's robust research going on
but that it's not,
that's not impeded
but that we also have
appropriate regulations
around this kind of very
powerful sort of application.
And you'll hear more
about that later this week
as Dana said.
And I'm gonna close just by mentioning
that all of this research
is done, of course, by
students and post-docs
in various laboratories
including our own
but we've had many many many
wonderful collaborations
and I'm just mentioning
a few of them here.
These are some that I mentioned today.
Karen Maxwell and Alan Jacobsen
at University of Toronto
are actively working on anti-CRISPRs
and we've had a great time
collaborating with them
as well as with Erik Sontheimer
at University of Massachusetts
and I mentioned these folks.
Science can't be done without funding
and I think we all have to be aware
that right now there's a very,
we're facing a very difficult situation,
especially here in the United States,
where there's an active push
by our federal government
to cut back on research funding,
especially for the kind
of fundamental research
that I talked about today
and so you can see that
if we really do that
I think we're gonna put the
US at a severe disadvantage
in terms of discovering new technologies
and moving ahead in the areas of research
that we know are gonna be important,
not only for human health
and our environment
but also for stimulating the economy,
creating jobs, et cetera.
So I would ask all of you
to think about ways
that you can communicate
the importance of the
science that you're doing
and encourage your friends,
neighbors, families,
who are, all of us are taxpayers,
to take appropriate measures
as they feel so moved
to support science funding
at a fundamental level.
And I'm gonna stop there.
I don't know if we have time for questions
but if you have any I'd be
happy to try to answer them.
Yeah.
(student asking question)
Yeah.
(student asking question)
It does, it hangs onto the DNA, yep.
I didn't have time to say
that but that's right.
It has a very high binding
affinity to the DNA
even after cutting.
(student speaking)
Yes, yep yep, you can do that
actually using this FRET
essay we can do that.
So there's a lot of nice,
I think a lot of nicer
biophysical measurements like that
that we can make.
Now one of the caveats with all of that
is that we're doing that
in vitro, of course,
we're not doing it in
the context of chromatin
so then we have to have other ways
that we can try to
relate those measurements
to what might be happening in cells
and there are already
we've done some of this
and there are other labs
looking at invivo imaging
of Cas9 so you can really watch behaviors
in the nucleus as well.
So I think over time we'll build
up this very nice continuum
between biochemical experiments in-vitro
and then things that are
going on in-vivo in the cell.
Yep.
(student speaking)
(laughter)
You can imagine there's probably, yeah,
anti anti-CRISPRS and yeah.
Sure, I mean, I think
it's sort of one of those things
where if you can imagine it
it's probably out there
somewhere in biology
and if it's not out there already
you can imagine ways
that you could potentially
engineer such things.
So I think one of the things
that's going on right now,
so there's been a big push
towards using
engineered proteins or natural ones
to control the activities
of Cas9 enzymes in cells
for purpose of safety
and also for the purposes of accuracy
and in fact Jacob Corn,
I think he just stepped out,
but his group with us
have recently been using
anti-CRISPRs for that purpose
where you can actually
limit activity in cells
to reduce the activities
that might be occurring
at off-target sites.
So think this is an exciting time
where we're seeing very
rapid technology development
around not the fundamental
core activity of Cas9
but really how we modulate
that activity in cells
using some of these types of approaches.
Yeah.
(student speaking)
Yeah yeah, that's a great question.
So there's a lot of work
going on on adaptation
so that means basically the acquisition
of new viral sequences
into these CRISPR arrays
and didn't have time to tell you about it,
out lab has done a lot on this
and there's other labs, of course,
contributing as well
but we know that there's a
particular CRISPR inner grace.
It's a two protein complex,
actually has six copies of those proteins
that form the inner grace
and that protein complex
is able to find and
integrate new bits of DNA
into the CRISPR array very precisely
by maintaining the structure of the array
and in some types of cells
like in e-Coli K12
there's an enzyme that,
it's actually not an enzyme,
it's a DNA binding protein
that bends DNA very sharply
and contributes to the
accuracy and efficiency
of that integration pathway.
So it's a great system.
George Church's lab has actually done work
using the inner grace as a technology.
So they're using it as a way
to do sort of cell recording
where they can integrate bits of DNA
into populations of cells
much as happens in nature
and use that as a way of
recording information.
So I think there's sort
of really interesting
forthcoming applications of other aspects
of these systems
that haven't been harnessed fully yet
but take advantage of
the natural mechanism
including the adaptation mechanism.
You should tell me when to stop.
Yeah, maybe one more and
then we'll take a break.
We can continue discussing outside.
(student speaking)
Yes, the one that I showed you does, yeah.
Yes.
It is effected actually, yeah,
because it turns out
that those two domains are highly coupled,
at least in the proteins
that we've been studying,
so when you inhibit one domain
even though you're blocking
just the active site
that cuts one of the DNA strands
it actually does affect
cutting of the other strand.
(light futuristic music)
