Hi my name is Jennifer Doudna from UC Berkeley
and I'm here today to tell you about how we uncovered
a new genome engineering technology.
This story starts with a bacterial immune system
that means understanding how bacteria
fight off a viral infection.
It turns out that a lot of bacteria
have in their chromosome,
which is what you are looking at here
a sequence of repeats shown in these black diamonds
that are interspaced with sequences
that are derived from viruses
and these have been noticed by microbiologists
who were sequencing bacterial genomes but nobody knew
what the function of these sequences might be
until it was noticed that they tend to also occur
with a series of genes that often encode proteins
that have homology to enzymes that do interesting things
like DNA repair.
So it was a hypothesis that this system
which came to be called CRISPR
which is an acronym for this type of repetitive locus
that these CRISPR systems could actually be
an acquired immune system in bacteria
that might allow sequences to be integrated
from viruses and then somehow used later
to protect the cell from an infection
with that same virus.
So this was an interesting hypothesis
and we got involved in studying this
in the mid 2000's right after the publication
of three papers that pointed out
the incorporation of viral sequences
into these genomic loci.
And so what emerged over the next several years
was that in fact these CRISPR systems
really are acquired immune systems in bacteria
so until this point no one knew that bacteria
could actually have a way to adapt
to viruses that get into the cell
but this is a way that they do it
and it involves detecting foreign DNA
that gets injected like shown in this example
from a virus that gets into the cell
the CRISPR system allows integration
of short pieces of those viral DNA molecules
into the CRISPR locus
and then in the second step
that is shown here as CRISPR RNA biogenesis
these CRISPR sequences are actually transcribed
in the cell into pieces of RNA
that are subsequently used together
with proteins encoded by the CAS genes
these CRISPR-associated genes
to form interfering or interference complexes
that can use the information in the form
of these RNA molecules to base pair
with matching sequences in viral DNA.
So a very nifty way that bacteria
have come up with to take their invaders
and turn the sequence information against them.
So in my own laboratory
we have been very interested for a long time
in understanding how RNA molecules
are used to help cells to figure out
how to regulate the expression of proteins
from the genome.
And so this seemed like also a very interesting
example of this and
we started studying the basic molecular mechanisms
by which this pathway operates.
And in 2011 I went to a scientific conference
and I met a colleague of mine,
Emmanuelle Charpentier who is shown in this picture
on the far left and Emmanuelle's lab
works on microbiology problems and they are
particularly interested in bacteria
that are human pathogens.
She was studying an organism called
Streptococcus pyogenes which is a bacterium
that can cause very severe infections in humans
and what was curious in this bug was that it
has a CRISPR system and in that organism
there was a single gene encoding a protein
known as Cas9
that had been shown genetically to be required
for function of the CRISPR system
in Streptococcus pyogenes,
but nobody knew at the time what the function
of that protein was.
And so we got together and recruited
people from our respective research labs
to start testing the function of Cas9.
So the key people in the project
are shown here in the photograph
in the center is Martin Jinek
who is a postdoctoral associate in my own lab
and next to him in the blue shirt
is Kryztof Chylinski who was a student
in Emmanuelle's lab
and so these two guys together with
Ines Fonfara who is on the far right,
a postdoc with Emmanuelle
began doing experiments across the Atlantic
and sharing their data.
And what they figured out was that
Cas9 is actually a fascinating protein
that has the ability to interact with DNA
and generate a double stranded break
in DNA at sequences that match
the sequence in a guide RNA
and this slide what you are seeing
is that the guide RNA
and the sequence of the guide in orange
that base pairs with one strand
of the double helical DNA
and very importantly this RNA
interacts with a second RNA molecule
called tracr that forms a structure
that recruits the Cas9 protein
so those two RNAs and a single protein
in nature are what are required
for this protein to recognize
what would normally be viral DNAs
in the cell and the protein
is able to cut these up,
literally by breaking up the double helical DNA.
And so when we figured this out
we thought: wouldn't it be amazing
if we could actually generate a simpler system
than nature has done
by linking together these two RNA molecules
to generate a system that would be a single protein
and a single guiding RNA.
So the idea was to basically take
these two RNAs that you see on the far side
of the slide and then basically link them together
to create what we call
a single guide RNA.
So Martin Jinek in the lab
made that construct
and we did a very simple experiment
to test whether we truly had
a programmable DNA cleaving enzyme
and the idea was to generate short single guide RNAs
that recognize different sites in a circular DNA molecule
that you see here
and the guide RNAs were designed
to recognize the sequences shown by the red bars
in the slide and the experiment was then
to take that plasmid, that circular DNA molecule
and incubate it with two different restriction (or cutting) enzymes,
one called SalI which cuts
the DNA sort of upstream at the far end
of the DNA in this picture
in the grey box,
and the second site being directed
by the RNA-guided Cas9
at these different sites shown in red.
And a very simple experiment
we did this incubation reaction
with plasmid DNA and this is the result
and so this is what you are looking at
is an agarose gel
that allows us to separate
the cleaved molecules of DNA
and what you can see is that in each of these reaction lanes
we get a different sized DNA molecule released
from this doubly digested plasmid
in which the size of the DNA
corresponds to cleavage at the different sites
directed by these guide RNA sequences
indicated in red
so this was a really exciting moment
actually a very simple experiment that was
kingd of an “A ha!” moment
when we said we really have a programmable DNA cutting enzyme
and that we can program it with a short piece of RNA
to cleave essentially any double stranded DNA sequence
so the reason we were so excited
about an enzyme that can be programmed
to generate double stranded DNA breaks
at any sequence is because
there was a long standing set of experiments
in the scientific community that showed
that cells have ways of repairing double stranded DNA breaks
that lead to changes
in the genomic information in DNA
so this is a slide that shows that
after a double stranded break is generated
by any kind of enzyme that might do this
including the Cas9 system
those double stranded breaks in a cell
are detected and repaired by two types of pathways
one on the left that involves
non-homologous end joining
which the ends of the DNA are chemically ligated
back together usually with introduction
of a small insertion or deletion
at the site of the break
and on the right hand side
is another way that repair occurs
through homology directed repair
in which a donor DNA molecule
that has sequences that match those
flanking the site of of the
double stranded break can be integrated
into the genome at the site
of the break to introduce new genetic information
into the genome
so this had given many scientists
the idea that if there were a tool
or a technology that allowed
scientists or researchers to introduce
double stranded breaks at targeted sites
in the DNA of a cell then together
with all of the genome sequencing data
that are now available we know the
whole genetic sequence of a cell
and if you knew where a mutation occurred
that causes a disease for example
you could actually use a technology like this
to introduce DNA that would fix a mutation
or generate a mutation
you might like to study in a research setting
so the power of this technology is
really the idea that we can now generate
these types of double stranded breaks
at sites that we choose as scientists
by programming Cas9 and then allow
the cell to make repairs that introduce
genomic changes at sites of these breaks
but the challenge was how to generate the breaks
in the first place and so a number
of different strategies had been produced
for doing this in different labs
most of them, and I'm going to show
two specific examples here
one called zinc finger nucleases
and the other TAL effector domains
these are both programmable ways
to generate double stranded breaks in DNA
that will rely on protein-based recognition
of DNA sequences so these are proteins
that are modular, and can be generated
in different combinations of modules
to recognize different DNA sequences
it works as a technology
but it requires a lot of protein engineering
to do so, and what is really exciting
about this CRISPR/Cas9 enzyme
is that it is a RNA programmed protein
so a single protein can be used for
any site of DNA where we
would like to generate a break
by simply changing the sequence
of the guide RNA associated with Cas9
so instead of relying on protein-based recognition
of DNA we're relying on
RNA-based recognition of DNA
as shown at the bottom so what this means
is that is just a system
that is simple enough to use
that anybody with basic molecular biology training
can take advantage of this system
to do genome engineering
and so this is a tool that really
I think, fills out an essential
and previously missing component
of what we could call biology's IT toolbox
that includes not only the ability
to sequence DNA and look
at its structure, we know about
the double helix since the 1950's
and then in the last few decades
it's been possible to use enzymes
like restriction enzymes
and the polymerase chain reaction
to isolate and amplify particular segments
of DNA and now with Cas9
we have a technology that enables
facile genome engineering
that is available to labs around the world
for experiments they might want to do
and so this is a summary of the technology
of the 2-component system
it relies on RNA-DNA base paring
for recognition
and very importantly because of the way
that this system works it
is actually quite straight forward
to do something called multiplexing
which means we can program Cas9
with multiple different guide RNAs
in the same cell to generate
multiple breaks and do things
like cut out large segments of a chromosome
and simply delete them in one experiment.
And so this has led to a real explosion
in the field of biology and genetics
with many labs around the world
adopting this technology
for all sorts of very interesting
and creative kinds of applications
and this is a slide
that's actually almost out of date now
but just to give you a sense
of the way that the field
has really taken off
so we published our original work on Cas9
in 2012 and up until that point
there was very little research
going on on CRISPR biology anywhere
it was a very small field
and then you can see that
starting in 2013 and extending
until now there has been this
incredible explosion in publications
from labs that are using
this as a genome engineering technology
so it's been really very exciting for me
as a basic scientist to see what started
as a fundamental research project
turned into a technology that turns out
to be very enabling for all sorts
of exciting experiments
and I just wanted to close by sharing
with you a few things
that are going on using this technology
so of course on the left hand side
lots of basic biology that can be done now
with the engineering of model organisms
and different kinds of cell lines
that are cultured in the laboratory
to study the behavior of cells
but also in biotechnology being able to
make targeted changes in plants
and various kinds of fungi that could be very
useful for different sorts of industrial applications
and then of course in biomedicine
with lots of interest in the potential
to use this technology as a tool
for really coming up with novel therapies
for human disease I think is something
that is very exciting and is really something
that is on the horizon already
and then this slide just really indicates
where I think we're going to see this going
in the future with a lot of interesting
and creative kinds of directions
that are coming along in different labs
both in academic research laboratories
but also increasingly in commercial labs
that are going to enable the use of this
technology for all sorts of applications
many of which we couldn't even have
imagined even two years ago.
So very exciting and I want to just acknowledge a great team
of people that have been involved in working
on the project with me and we've
had terrific financial support from various groups
as well and it's been a pleasure
to share this with you, thank you.