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