(applause)
>> Yeah.
All right.
Well, thanks
for coming.
Some of my students are here,
and that's just voluntary,
which is amazing.
So...
So I wanna thank Tim for
actually putting these together.
You know, you get used to
kinda doing the same thing
over and
over again.
And you-- and I think it's neat
to actually have something
to talk about that--
I'm not a CRISPR scientist.
I will tell you a little
bit about what I do.
But reading about it
over the last past year
and doing honors
projects with students
has really given me
an opportunity
to just kind of
expand my mind,
which, without people like
Tim pushing us constantly,
like, almost annoyingly...
(laughing)
even when I was
sitting up here,
he was trying to get
another talk next year.
So, but anyways,
I think it's good.
I think it's good for
all of us to be thinking
about new things.
So first of all, why am I
interested in this topic?
Well, when I was
in graduate school,
one of the things
I worked on was
I did work on DNA arrays
for different organisms.
And I looked at
recognition for DNA.
And one of the things that
we'll talk about today
with CRISPR is that
one of the things
that makes it unique
is it identifies
unique sequences
of DNA.
And so, I have a past background
of doing amplification
and recognition of
big biomolecules,
and then trying to analyze
them in the gas phase.
So we were doing more
diagnostic components with DNA.
I've worked a little
bit with some students
and faculty out at Hope
looking at doing mutations
in genes and seeing how those
ultimately affect an organism,
and so, protein
modifications.
And then, I have a
couple students here
that did honors projects
in reading this book.
I found this a
couple years ago--
it's "A Crack
in Creation."
I know a few of
you have read this,
and it is about scientist
Jennifer Doudna
out at Berkeley that
helped develop this tool.
And she's an
RNA chemist.
And so, it's not like
she knows bacteria
or different things.
She looks at structures
of RNA molecules.
So, and I just think it's
kind of an interesting story.
So if you read what I
wanted to cover today,
it was really looking at
how this process came about,
and then talking
about, like,
"What are some of
the applications?"
And so, you can
ask me questions,
you can comment
at the end.
Maybe you know something
I don't know, I hope.
Or you can say, like, that was
the most ridiculous thing.
This is
actually true.
So-- but I'm gonna kinda take
you through this process.
And you-- this has been
showing up in our news
over and over again, this
concept of gene modification
and CRISPR.
And some of the things
that goes on in science
is when a new technique
is developed,
there are patent
disputes.
And so, you look at some of
the high-power researchers
in CRISPR out at
Berkeley or MIT,
and they fight over the
uses of these things.
And it's because these have
strong diagnostic tools.
That means money's
gonna probably be made.
And so, modifications
around this.
So people are kinda vying
for patents around this.
So you see it in the
news around patents.
If you were at the end
of the year last year,
you might've seen that
a scientist in China
had modified some embryos so
that they were HIV-resistant.
And so, I'll talk about
that a little bit.
That's a little
bit controversial.
And then, like I said,
with patents come stocks.
And you will-- I know some
people that have owned these,
and it's an emotional
roller coaster
if you own any stock
around CRISPR,
because it goes up and
down with FDA approvals.
And so, we'll talk about some
of the limitations of it, too.
And I'm not gonna give
you stock tips, though.
But what I'd like--
so the outline
for my presentation is
I am gonna give you
a quick overview
of biochemistry.
And it's just so that when
I talk about this system
and I talk about nucleic
acids or amino acids,
you know what I'm-- you can at
least reference back to this.
And if you do
wanna copy this,
I'm happy to
send this to you,
'cause I know probably
in your pastime,
that's what you wanna
review, is this talk.
(chuckling)
But I will say
that I have sat through
many of these.
My hope is that you take like
one or two things home with you.
And I'll try
and be short,
'cause everybody loves
when these get out early,
and you get a cookie,
it's great.
So I am gonna talk a
little bit about bacteria.
I'm not a biologist,
but-- and bacteriophage.
A few definitions,
because I think
you can get caught up
in the terminology.
Adaptive immune system,
I'll explain that
when we
get to it.
And then, what are some
of these applications
of this CRISPR-Cas9
system?
And it's
fascinating.
And I am gonna talk about a
lot of the limitations, too,
because I
don't-- I--
it's like any
drug delivery.
Getting it to
cells is tricky.
And so, we'll
chat about that.
So you know, if you've taken
a general biology class
or a chemistry class
before, that DNA--
if you ask even a
little kid what DNA is,
they'll say it's what
makes us "us," right?
And we know that from
a genetic perspective,
DNA codes for genes,
right, and those genes
can be transcribed
to RNA,
which is a compound that
can ultimately be used
and translated into
proteins, okay?
And proteins are what carry
oxygen around our blood,
they break down hydrogen
peroxide in our body.
All the systems
that you've learned
around glycolysis
and the Krebs cycle,
they all have a
component in them.
So I'm gonna talk about
DNA and RNA and proteins,
'cause they're all involved
in this process, okay?
So nucleic acids, that's
where we're gonna start.
And we have to have
some molecules up here,
because when you
think of DNA,
that's what you're
used to, right?
Seeing the double helix and the
kind of two rungs of the DNA.
But you know that it is made
up of those four bases--
adenine, thymine,
guanine, and cytosine.
And they pair up
really specifically.
And that's sort of the
crux of this technique
is something has to make
sure that they pair up,
when DNA pairs up
with each other.
When we
recognize DNA,
it's because of those As
and Ts and Cs and Gs, okay?
Nucleic acids can be in
the form of RNA, as well.
So DNA gets
coded to RNA.
And usually we think of
this as single-stranded.
But it's way more
complex than that.
The RNA folds up into
proteins-- or excuse me,
folds up into structures
like this one right here,
which certainly looks like it's
paired up with one another.
And it's why Professor Doudna
actually got involved in this,
because there is a lot
of RNA kind of super--
that secondary-type
structure there, okay?
So nucleic acids,
RNA, and DNA.
And then, when you
think about proteins,
and I'm gonna stop with the
biochemistry here in a minute,
amino acid chains
are proteins.
So that DNA codes for RNA,
which codes for a protein.
And proteins are made up
of these simple little
amino acids,
long chains of them.
In fact, the Cas9 system,
the gene editing tool
that I'm gonna
talk about,
actually has about a
little over 1,300 of these,
so 1,300 of these
strung in a row.
And so, that gets untenable
from a chemicals perspective.
It's hard to, say,
show you 1,300 times 10
or 130,000,
140,000 atoms.
So we use
cartoons.
And you've probably
seen those before,
kinda those
alpha helices
and those sheets,
those beta sheets.
And the structures that
I'm gonna show you
are gonna be more
like cartoons
than they are gonna
be like, sadly,
than like atoms strung
together, okay?
Again, it's why
people love biology,
and then they
come to chemistry
and they see this,
and they're like,
"Can we do
this again?"
So here's a couple
protein structures.
And you know
they're cartoons!
They're cartoons of
those alpha helices
and those
beta sheets.
This is
hemoglobin.
It's kind of a
low-resolution picture.
This is a beautiful
complex between a protein
and a piece
of DNA.
And you can see the
characteristic double helix
right across the top, and
then the protein over it.
And I'll come back to
that structure again here.
I'll actually come
back to both of these.
So there is-- so we know
we have our biomolecules,
our big biomolecules of
RNA and DNA and proteins.
But this story sort of
begins with bacteria.
And over 20 years ago,
I thought this
was an interesting
statement about bacteria.
It says they don't
have easy lives, right?
So we eat them, and we break
them down in our immune system,
and I read that half the
bacteria die every day,
every couple days.
And I've talked to a
few of you about that.
I'm not sure
it's true.
But I like the idea of it,
that they're under attack.
The point isn't whether or not
that's statistically correct,
'cause some are growing faster
and some are growing slower.
The fact is bacteria
are under attack, right?
They're under
attack by our guts,
as well as these
viruses that exist
just to break them apart
and use them, really.
So when you think about
a virus, a bacteriophage,
they land on the
surface of a bacteria.
And they kinda look like
that cool land rover, right?
Right here.
And here's a nice little
structure of them.
And they-- if you know
anything about a virus,
they insert their DNA
into that bacteria.
And then, they use it to make
more of themselves, right?
And then, they lyse open
and take advantage of it.
So you know that the cell
fate when a virus is on it
is going to be
death, right?
It's going to use it,
it's gonna burst it open,
and spew out a bunch
of itself again.
And so, what has happened
over years of evolution
is it's developed a system
to combat these things
when they come
onto their surface
and inject
their DNA.
And that's where
CRISPR comes in.
What I like about this is
it was over 20 years ago
when it was first identified,
this CRISPR system.
And so, I have-- I'm gonna
use pool noodles to do this,
because it's
a visual.
And that's the only way I can
really think about this system.
And Tim's gonna help
me here in a minute.
But if you think about
this as a piece of DNA--
and it's a cartoon,
obviously, here.
But if you think of
these as kind of genes
and these little segments where
you can see there's purple
and a color and purple
and a color and purple.
And in science, you know that
we look for patterns, right?
And so, back in 1987,
it was identified
that this pattern
was here.
But then, it took
20 years to figure out
that it is actually part of an
adaptive immune system, okay?
The adaptive immune system
of these bacteria.
And so, we'll get into how
this actually happens here.
But when you think
of CRISPR, I never--
I was a
LASER chemist
and I always forgot
what "LASER" stood for.
You know, it was
"Light Amplification
"of Stimulated Emission
of Radiation."
You could never-- I
never remembered that.
I never remember what
CRISPR stands for.
But the words
"Clustered," right?
Close together.
"Regularly" means they
have a specific size
associated
with them.
"Interspaced, Short--"
not long, and that's relative.
I'll show
you that.
And then, a "Palindrome"
sequence in biology
and chemistry
matters.
So it's the same
forwards and backwards.
And what that does is
it allows DNA or RNA
to have a specific
sequence.
They'll pair
with themselves
and make hairpin structures
because of it.
And I'll show you
a few of those.
And then, "Repeats," over
and over and over again.
And I hope someone
at the end asks me
how many times does it
repeat over and over again,
because I'm ready
for that one.
So we're gonna chat a little
bit about what these are.
So that's what
CRISPR stands for.
That basically is recognizing
this pattern down here,
this repeated "repeat spacer,
repeat spacer" sequence.
Now, when we talk
about Cas genes--
so this is kind
of a hybrid,
this system that I'm gonna
talk about is a system hybrid
between proteins
and nucleic acids,
so DNA, RNA,
and proteins.
So something has to play
the role of these genes,
the proteins, everything
when they interact
with viral DNA or the DNA
within the bacteria,
it's usually a
protein with it.
So I'm gonna play that
role as the protein.
I actually specifically
wore "Cas" here
so that you can see this,
the Cas protein.
I thought to myself
this morning
that I ruined a
perfectly good t-shirt,
unless someone goes--
'cause I'll wear it,
and someone will go, "They
added a C and forgot an S,"
you know, when--
(audience laughing)
so, but I'm going to--
I'll be--
whenever we're
talking about proteins,
I'm gonna play
that role.
I'm gonna be that
amino acid chain.
And these Cas proteins do
lots of different things.
So they are nucleases,
which means they cut up DNA.
They separate, because
if you're gonna deal
with viral DNA
that's coming in,
you have to be able
to separate it apart.
And then, they
do other things,
like identify unique
sequences, as well.
So whenever I'm holding the DNA,
remember, I'm the protein, okay?
So let's get into
what this means.
So I'm a
bacteria.
Let's say the stage is
the bacteria up here.
And I'm gonna be invaded
by a particular virus.
And Tim's gonna
be that virus.
He's gonna be carrying
the DNA in here.
And there are basically
three fundamental stages
of this bacterial
adaptive immune system.
So they wanna get ready so that
when a virus attacks them,
they know what
they're going to do.
They'd like to destroy
that viral DNA
before it actually takes over
and destroys that bacteria.
So the first stage of this
adaptive immune system
is actually taking
a piece of this DNA
out of this virus
that's coming up.
So Tim, if
you'd come up?
What happens is when he brings--
oh, he already did it.
Yeah.
(chuckling)
So-- yeah, and it happens--
there's a mechanism--
that's all you have to
do-- I appreciate it.
I'm gonna call you
back up for more.
(audience laughing)
You did your job.
So I, as a Cas protein
inside of this bacteria,
I am--
like Cas1 and 2,
I'm gonna take this,
when it gets inserted
into the bacteria through
the cell membrane.
And I'm gonna look for
the specific spot on this.
And you'll notice that I
have this black line here.
I'll explain
what it is.
When I recognize that, I'm
actually gonna clip out
a little piece of DNA from
that viral DNA, okay?
And then, that obviously
gets cut up and it's gone.
And I'm gonna insert it on
the end of my CRISPR array.
So remember, this is the
normal bacterial genes
that code for, like, me,
the Cas gene,
the things that are
involved in this system.
And then, you'll notice
it's been exposed
to a couple
viruses.
And if you don't mind
holding those up.
Like, those might be
DNA from other viruses,
like a red piece, a
little turquoise-y piece,
and now this yellow piece that
I'm gonna add to this, okay?
So I've taken a piece
of DNA from a virus
and incorporated it
into my own DNA.
I'm a bacteria,
right?
So now, I have this
CRISPR system that has--
I've just added a
new repeat to it.
So it's got this repeated
pattern, a spacer...
that's what type
of DNA now?
>> (indistinct).
>> Viral DNA.
These different
viral DNA.
I've actually incorporated
it into my own DNA.
So it matches the sequence
of other viruses, right?
So this yellow,
if I am exposed
to another yellow piece of DNA,
I have a matching sequence.
So I have these genes and
these repeated sequences.
They're not
very long.
And this actually is kind of a
key to how this system works.
You notice they're about
20 nucleotides long.
And that's gonna
come into play.
I'm gonna talk about
that here again later,
because it has to
do with statistics.
The longer the
piece you come in,
the more specific
it would be,
because, you know,
if everything has an A,
then an A and a T,
there's less likely,
and then A, T, C, statistically
it becomes less likely
to come across a
longer pattern.
So they incorporate
this 20--
about 20 nucleotides, and then,
this other little piece of DNA
that they use over
and over again,
that black piece, that's
about 30 nucleotides.
So a total of 50 in
this CRISPR array, okay?
You'll notice it says
Cas1 and 2 there.
This is the Cas1
and 2 system.
So bacteria
have this gene,
like those Cas genes make
this particular protein.
And it's pretty
complex.
It's got, like, lots
of amino acids in it.
It's a pretty
big protein.
But the distance between
it actually determines--
that's what I think
is kinda elegant,
it keeps cutting
the exact same size
of piece of DNA
because of its length,
the protein
length of it.
So it binds
to viral DNA.
This would be
the virus.
And it clips that
little piece of DNA
and adds it to its
CRISPR sequence, okay?
So DNA, according
to our central dogma,
becomes RNA,
right?
So DNA codes
for RNA.
So what the bacteria does
is it turns this sequence now
into little
pieces of RNA.
And this is called
"pre-CRISPR RNA."
And you'll notice it
has different colors,
and it has that little hairpin
sequence, as well, okay?
So what it does is
it takes this off,
an enzyme would come along and
make this piece of RNA, okay?
And then, it would break
it up into little pieces
like this, so it
expresses these.
And then, there'd need
to be more of me.
But I would hold
these in my hand.
So I am ready for these viruses
to come in-- into the--
so I'm gonna ask Tim to
come up with a new one here.
So if you think of
this system now,
I have this-- I'm a
protein with this one,
I'm a protein with this one,
or I'm a protein with this one.
It looks like he's coming
in with some red here.
So what I'm gonna do
with this Cas9 system
is I'm gonna come up
and I'm gonna go,
"All right, this
matches, right?"
I'm gonna look for
that black line.
It matches.
And I'm gonna
cut it.
What happens to the
viral DNA when it's cut?
It doesn't
work anymore!
So now, that virus
is no good anymore.
So I'm gonna bring
the other one up.
I am this Cas9 system
with this piece of DNA.
I come up, I look
for this sequence,
I match it up, and
then I cut it.
And now, that virus
is no good anymore.
So I am just, like, a
virus-destroying machine,
as long as I've seen
it already, right?
So if I've seen it, I've
incorporated it into my DNA,
I make the RNA from it, and
then I have this protein
that, when this virus
inserts a piece of DNA,
this is the
Cas9 system.
It recognizes it.
This PAM sequence
is that little mark
that I'm gonna
talk about.
And I bind to it, I make
sure that it matches,
and then
I cut it.
And when you
cut viral DNA,
you make it so that it
can't use the machinery
in the bacteria to make
more of itself anymore.
So that's all the
CRISPR system is doing.
It's taking, incorporating
a short piece of DNA
from a virus into
its own genome,
it's turning it into RNA,
and turning it into a system
that recognizes DNA in foreign,
invading bodies, okay?
So I keep talking--
and you'll notice--
this is actually an
important thing.
So when you leave and if you
ever wanna read about this
before, you'll notice
there's some limitations
to this system, is
there always has to be
this little
black mark.
And this is usually a
three-nucleotide sequence
that me, as a Cas enzyme, has
to recognize before I'll cut.
Now, this is actually
pretty important,
because if
you'll notice--
and it doesn't matter
the order of this.
This worked so well in my mind.
(laughing)
So if you look at
this sequence here...
Let's put
that one on.
Oh, that's
the problem.
Hang on.
We can edit
this out.
If you think of
this sequence here,
the difference between it
is this piece of viral DNA
has that black
line on it.
My DNA inside
the bacteria
does not have
that black line.
And so, you've heard
of autoimmune diseases
where your own immune
system attacks, right,
your own cells.
What this little
sequence--
it's called the "protospacer
adjacent motif"--
and it's three nucleotides
that the Cas gene-- me--
looks for before
I'll cut.
So I would go along my
own DNA as a bacteria,
and I'd go, "Oh, my gosh,
that sequence looks like
"what I wanna cut."
But it won't, because
it doesn't have
that little "PAM sequence,"
they call it,
the "Protospacer
Adjacent Motif."
So you will see this if you
read about the CRISPR system.
That's kind of
a unique thing,
and it ends up
being a limitation,
because if you
wanna cut DNA later,
you can only cut when you
have a unique sequence,
like any random
DNA, okay?
So it's really a
self-recognition tool
for this
system.
By the way, finding
pool noodles in February
in Michigan...
(chuckling)
it's probably like trying
to buy a sled in July.
I think they probably are
in the same warehouse...
especially when you want
the same colors, too.
So they have to recognize
that PAM sequence
before they'll
cut, okay?
So back to--
so that's virally,
and from a bacterial
perspective, what happens.
Bacteria clip out a little
piece of DNA, save it.
When they see something just
like it, they cut it, okay?
Where this system starts
to get interesting
is in order for
this Cas9 system,
this protein
to function,
it needs
CRISPR RNA.
So it needs this.
It needs a sequence that
matches a virus, or anything,
this kind of random spacer
that forms that loop,
and then it also needs
another piece of RNA
that they don't really
know exactly what it does,
but they know that it
helps prop up the protein,
like, it's a
structural thing.
And I'm gonna show you
an image of it here.
This is an image I found
from that Doudna lab
where it's this
Cas9 system.
And what Berkeley
is famous for
is they took and made this
into one piece of RNA.
So instead of needing this
and another piece of RNA
for this system to work,
they linked them together.
And they made what's
called a "guide RNA."
So when you think of it
from an application of,
"How are we gonna
modify some DNA,"
they call it a
"single guide" or "sgRNA."
And it includes
the piece that goes
and recognizes
the virus,
and then this other piece
that's a scaffold
or a structural
piece of RNA
that seems to need
to be there, okay?
'Cause if you take it
out, it won't work.
So what scientists
did at Berkeley
was they made this
one piece of DNA.
And they have a patent on
that mechanism, okay?
So I wanted to show you--
the protein database
is kind of a neat thing
if you've ever done
any DNA or RNA or
protein research.
I found this on
their website.
And I just wanna
show it to you,
'cause it gets this protein
three-dimensionally.
And I was sort of amazed they
had put some music to it,
which I
thought was--
I don't know, I thought
it was kinda interesting.
So just enjoy
looking at the Cas9.
This is the Cas9 system,
which CRISPR,
which we know is the array,
and the Cas9 protein.
This is the system that goes
and finds and cuts DNA.
So...
(tranquil piano music)
This goes on for 30 more
minutes, is that okay?
(audience laughing)
But you can see, it's
sort of an elegant system.
It's got this
protein, again,
and you can see the
different components to it.
It's a pretty complex protein
that binds to DNA.
You saw the pieces
of DNA coming in.
It cuts it,
okay?
That's what renders
the viruses inactive.
And that's a big deal, because
if you think about this, um...
Oh, let me go
to "So What?"
You have a device in a bacteria,
but you could pull it out,
and you could say,
"Well, I have this tool
"that can recognize
sequences of DNA.
"20 base pairs is
pretty unique, okay?
"And it's a
snipping tool
"so it can recognize
and cut DNA."
So when people say the
CRISPR-Cas9 system,
that's what they're
talking about,
a piece of-- well,
a bacterial protein
that can recognize, with
a piece of RNA in it,
that can recognize
and cut other DNA.
So that's what
we're talking about.
And I like that from,
like, Word 2000.
So what happens
when you cut DNA?
So why do we care
about this?
So when you cut DNA, a
couple things can happen
in eukaryotic
cells.
If you just cut it,
it sorta scrambles.
If there's nothing that
it can match off of,
what happens is it will--
they call them "indels,"
where they have insertions
and deletions into the DNA.
So if you have a gene
that, let's say,
it is coding for a protein
that you don't want it
to code for
anymore.
If you go in with a
CRISPR-Cas9 system and cut it,
it's gonna screw up all
those bases in that DNA.
And it's gonna make it so the
protein doesn't exist anymore,
the protein doesn't
work anymore.
But even more interesting is
if you put in this Cas9 system
to cut a piece
of DNA
and you have a piece
of DNA that's correct,
that has a
difference in it,
what it will do is
it'll go through
homologous
directed repair
and actually make a new piece
of DNA that's right or correct.
So it's a way of correcting
problems in the DNA, okay?
And I'm gonna give you a
couple examples of this.
If you've taken a biology
class or you've learned
about sickle cell
anemia, okay?
Sickle cell anemia,
if you look over here--
and that was why I
started with RNA to DNA.
This is a normal
gene for DNA
for someone without
sickle cell anemia.
And if you notice,
it says G-A-G.
If you have sickle cell,
you don't have G-A-G.
You have G-T-G,
okay?
It's a one base pair
difference.
So if I could design
a CRISPR system
that recognized an area
around here and cut it,
then I had a template
that I put in with it,
it would do exactly
what this does.
So it would cut it, it would
find a new piece of DNA
with the corrected base,
and it would fix it.
Now, I put in this slide,
one of the things
they've tried
to do is--
"heterozygous" means you
have one correct pair
and one
incorrect pair.
So they have shown
that in certain cells,
they can cut it, and it will
use the other correct allele--
or excuse me, the
correct piece of DNA.
So we have two copies of
DNA, right, humans do.
It cuts
the bad one,
and it will use the
corrected one to make it--
to fix it,
in a sense.
So it'll switch
that T back to an A.
And then, hopefully,
the central dogma
is DNA becomes RNA
becomes protein.
Hopefully now
that protein
doesn't have that
weird amino acid.
And it's not weird,
it just doesn't have
the same charge on it.
And so, you get
those sickle cells.
And it's a
horrible disease.
So modifying DNA in
a eukaryotic cell
would be a
big thing.
If you can cut it,
it would repair itself.
Another example-- and this
has actually been done
in what's called
an "organoid."
Tim and I have worked a
little with a researcher
over at MSU.
He does some
CRISPR research.
And I visited his lab to see
how he was doing this work.
And they build--
they take cells
and they turn them
into stem cells
and grow little organs
in a Petri dish, okay?
And that's
really neat.
But what they can do is, if
you have cystic fibrosis,
you will often-- one of
the most common ways
you have cystic
fibrosis
is you have a three base
pair deletion in your DNA.
So you're missing-- I
don't remember what it is--
A-A-A,
I think it is.
But if you're missing
that, you could go in
and cut that region,
have a corrected piece,
and it would
fix that.
And it's just a
teeny little deletion,
but it makes a
huge difference.
If you know anybody
that has CF,
it's based on this
membrane protein
that helps regulate
water and chloride ions
in and out
of the cell.
And they get thicker mucus
and it clogs those pores.
And a devastating
disease that could--
if you could get a
CRISPR system in
to cut that DNA with
the correct template,
you could
fix that.
This is another
example of non-human,
but where they would have--
they have done this, actually.
They haven't released
these, I'm sure--
can you imagine
doing bug research?
Like flying bug
research?
You know they're out, right?
(laughing)
It would be-- I just
think that would be
an interesting
lab to visit,
how they contain
all the mosquitoes.
But what they do is they
do gene modifications
to make the females have
more male characteristics.
And so, their sexual
organs are deformed,
and their mouths
are deformed.
And these are specific
mosquitoes that carry malaria.
And so, if they release these
out into the wild, right--
I like this-- I just like
this image of mosquitoes
deciding they're gonna
pass their genes along.
And they call it
a "gene drive,"
where they have
baby mosquitoes,
and those baby mosquitoes all
have these deformed components.
And so, they don't
pass malaria.
And that might not
be a big deal to us,
because we don't live with that
many mosquito-borne illnesses.
But in a country where
there's a lot of malaria,
this would
matter, right?
There are two other
types of systems
that could potentially
be used here.
It's called
"CRISPR interference."
So what they do is they
take this Cas9 protein,
and remember, it
recognizes pieces of DNA.
And they can take this, and
they can lay it on the DNA
and then just have
it adhere there.
So they turn off the part
of it that cuts the DNA.
So all they do is use it
as a recognition tool.
And you can change
the structure of it
to make it bind
more tightly.
And it can go and
it can interfere,
and basically stop genes
from being expressed.
So you don't go from
DNA to RNA anymore,
'cause there's this stinking
Cas9 protein bonded to it.
So you can up-- you can upreg--
you can downregulate genes.
And then, they can
also upregulate genes,
where they use the
same CRISPR system,
they shut off the
ability for it to cut,
but they go and they
bind to a specific region
of DNA with other
components on it.
So they can modify the
Cas to attract things
that will make the
gene upregulate,
so make more
genes be created.
And they've
done this.
They've done this
on some organisms,
and they've done this
on mice to increase
the muscle mass.
So they turn on the genes
that make them build
more and more
muscle.
So from a food
distribution perspective,
that might be
beneficial, right?
Your organisms
create more meat,
if that's the type
of thing you're into,
and you could
feed more people.
So why aren't we seeing
this, like, everywhere?
I think that the
main component--
and I've read a lot about this,
to how you get this into cells.
So think about what
you have to deliver.
Me, a Cas protein,
you have to deliver me.
You wouldn't wanna deliver
a Cas piece of DNA,
'cause you don't
want a bunch of me.
I'll keep
cutting DNA.
So you don't
want me in there.
So you wanna
deliver a protein
and then that single
guide piece of RNA, right?
So those are the
two things you need
to recognize and cut DNA, and
possibly a template, right?
The correct version.
So if I'm homozygous,
I have a correct version,
but if I'm not,
I need to deliver
all these things to cells
to make this work.
So they've thought
about using viruses,
human viruses,
to deliver this.
But those typically
deliver DNA or RNA.
But there is sort of
some promising work
around these
exosomes.
And they're basically
lipid protein--
or excuse me, lipid
membranes that,
in this little packet
in the middle,
they could put
a protein.
They could put a piece
of RNA in there.
And then, it would
kind of assemble
when it made it
to the cell.
Easier said than
done, though,
because what
happens?
Our body mounts immune
responses to these things.
So it's tricky.
When I think of this
happening, like,
"Oh, this is gonna
solve cystic fibrosis
"and sickle cell anemia and all
the other genetic diseases,"
the hardest thing is getting
it to the cell, okay?
I have read a couple
promising things.
If you could
remove the cells
and then put them back in, like,
that has some real promise,
because if you
can take the cells
out of a human,
and modify them,
and put them back in,
that's a little easier.
But again, you don't want
Cas9 in there constantly.
It's just gonna continue to
cut DNA over and over again.
So I opened with that slide
on that Chinese scientist
that modified
embryos.
That's pretty
controversial, right?
Like, to modify a
undeveloped organism...
we probably all could have
an argument about that,
whether or not that
should be done,
because we might say, "Well,
all we're gonna use this for
"is to get rid of CF and
sickle cell anemia."
But you might say,
"Well, I want my offspring
"to have big arms or
strong arms or whatever,
"whatever,
blue eyes."
You might-- that might get
a little more complex.
But you get that that's the
best place to do it, right?
Because you've got one
cell you have to modify
instead of--
I mean, I've read--
they use the analogy
in this book, like,
"When do you
edit the paper?"
Before it goes
to print, right?
You edit the paper
before it goes to print
because once you, if
you modify one cell,
that's fantastic.
But once it has
produced a baby,
it is millions and
billions of cells.
So it's a little more
difficult to do.
So the differences
between germline cells,
egg and sperm, or somatic
cells-- skin, organs.
It's a little less
controversial
because you
don't have to--
you know, you're not gonna
pass that on to offspring,
but it's way
more difficult.
There also is, if you're
reading about this
a little bit more, DNA repair
can be pretty unpredictable.
So it's beautiful that
I showed those two slides,
like you get insertions
and deletions,
and you get a correction if
you have a correct template.
But it doesn't
always work that way.
That Chinese scientist,
what he did was...
there is a protein that HIV
will bind to on the cells,
our immune cells.
And you know, there's a
certain amount of people
in our population that
are immune to HIV.
You can be exposed to it,
and they have a mutation
on that protein
in their membranes
so the HIV virus
can't bind to it.
So what he did was he
knocked out that P,
he modified that gene so that
they have a modified protein.
Now, that's problematic because
that mutation happened
probably millions
of years ago.
And we have other genes--
we don't know a lot
about that gene
that HIV connects to.
So if it modified just
through the natural
process of evolution,
if other genes
took its place,
it if was
important,
other genes actually
compensate for it being mutated.
And so, when you just go in
and blindly modify one protein
and you don't know
a lot about it,
there could be other
consequences for those children
that were born that we
don't know about, okay?
So it is pretty controversial
for a reason, right?
So kind of in
conclusion,
this is an adaptive
bacterial immune system.
Bacteria are exposed
to viruses,
they clip out little
pieces of DNA,
and then they create
a protein-RNA system
that can recognize
and cut DNA.
We know that DNA
can repair itself
if there's
a template.
So cutting it,
there's a chance
that it could fix
little mutations.
I think a lot of the
interesting research
around binding without
cutting is really important,
because we can upregulate genes
and downregulate genes.
But right now, the
biggest issue, I think,
is delivering
this to cells.
And so, that's why
egg and sperm cells,
pretty easy
to access.
But if we wanted to go in
and modify my lung cells
because I had CF, that might
be a little trickier to do.
So that is-- I will give you
a little recommendation.
If you wanna read
a pretty good book,
Jennifer Doudna,
again, at Berkeley,
RNA chemist,
biologist, chemist...
we're all on the
same team, right?
They, uh--
she has a--
it kind of takes this really
good perspective on this,
because she looks at
the power of this
and worries about it from
an ethics standpoint.
And I think she's trying
to get in front of it,
as a community,
right?
And just because if
we were to pass laws
or make regulations
on this,
it's a big deal if we
do it in our country.
Other countries
could do it, right?
And so, science
isn't limited,
we don't limit science
based on our laws
in one country.
But she just wants to
open the conversation.
I think she does a nice job
kind of outlining this process
and then talking about
what it could become.
Tim mentioned-- well,
actually, Taylor mentioned
that sometimes we
think about this
in terms of, like, if we
were to make a designer baby
or something
like that,
only people with money would be
able to do something like that.
Where, in turn, she
makes the argument
in her book that we maybe
can't afford NOT to do this,
because if you could
cure a disease
or fix something before
it becomes a problem,
that might be cheaper
in the long run.
And so, there are a lot
of ethics around it.
But it's kind of
a fascinating topic,
and I appreciate Tim for letting
me come and talk about it.
And I'll take any
questions you have.
So...
(applause)
I think I got done-- yeah,
it's good, good timing.
>> I have a microphone.
>> All right!
When I interviewed for
my job 20 years ago,
Tom asked a pretty
mean question,
I'm not gonna lie.
(laughing)
>> Now, I'm gonna show
my ignorance here.
So when a bacteria
reproduces, replicates,
I don't know how
bacteria do this,
does it pass
on its DNA
that it's been changing
all along here?
>> Yeah.
>> So isn't that getting, like,
ungainly large after
it keeps getting--
>> Ah, that's a good question--
it's a good way to ask this.
So interestingly, this
is all over the place.
There are, like, dozens and
dozens of CRISPR systems.
Like, there's type 1,
type 2, type 3.
Some of them
recognize RNA.
A typical bacteria
only has--
that's a pretty
dynamic thing.
So they might only have
three to five CRISPR arrays.
I mean, they might not have
many incorporated viral DNA
in their DNA.
So they pass on
what they have
when they replicate
their own DNA.
But I have also read
that other organisms
can have hundreds of
spacers in there, yeah.
So yeah, but
there are limits.
So if you look
at the rate--
it depends on what they're
being exposed to, as well.
So it actually runs
the other way.
You have the Cas genes, and
they add in the middle...
and the things that
get further down,
they sooner or later
get clipped off
because they're not
being used as much.
So it can
turn it over.
It's not like once it's
in the CRISPR array,
it's always there.
So they do clip them
out after a while.
So, that's a
good question.
I like that one--
I knew the answer.
You redeemed yourself
after 20 years.
(laughing)
>> Any other
questions?
>> Could I ask you to
comment a little further
on some of the
ethical issues
that Jennifer Doudna
brings up in the book?
I can give a specific
prompt, if you like.
>> Oh, go ahead.
>> Yeah, yeah.
I was curious if
there's any talk
about who should have the
right to control gene editing.
Of course,
'cause you'd ask,
do you wanna hand it
off to the government?
Do you want patented
gene sequences
or gene editing
tools, like--
what is-- does she have
any commentary on this?
>> She does.
Primarily what-- she does talk
a lot about access to it.
And again, from--
it is more that--
so initially, she was a
little horrified by it
because she thought of what
could potentially come about
from this
technique.
But then, she got to this
place where she thought,
"If we can do this,
why don't we?"
Because it is
sort of about...
easing human
suffering.
And so, she comments
mostly on that.
She doesn't get--
obviously, there's economic
and different
components to it.
And she talks
about that, too.
But it's primarily
on basically costs.
And she-- I mean, I think
she's trying to get people
to talk about this from
the scientific community,
because it
is tricky.
Who does
run it?
Once people have
patents on this,
they have a patent, and so...
(chuckling)
and I think that
that's why she's kinda
having the discussion.
'Cause right now, there
are private companies
that own these
patents.
I don't know if I
answered your question.
It's pretty big.
>> (indistinct).
>> Okay, yeah.
>> (indistinct).
>> Yeah?
>> I have a question
pertaining to cancer.
Did you-- in your readings,
did you come across
any researchers who
were trying to insert
the sequence that will
allow cancer cells
to remember that they need
to go into apoptosis?
>> Yeah, they have a lot
of hopes with cancer cells.
In fact, over at
Michigan State,
that's one of the
interesting things,
is there's a common
mutation in cancer cells.
It's a tumor suppressor
gene that gets mutated.
And apparently,
the cell type
that you can
do CRISPR in
matters whether it's a
cancer or non-cancer cell.
It's easier to do it
when they're cancer cells
or when they have a
particular mutation,
which makes
some sense.
But absolutely, those
are the things that,
if you can sequence it and
find out where the errors are,
those are absolutely techniques
that they could use.
Like, turn on that
process again,
because something
has been mutated
that it's not getting that
signal to destroy itself.
Yeah, for sure.
>> So I know you brought
up autoimmune diseases
at the beginning
of the talk,
but I just wanted to ask you to
expand on that a little bit.
What are the hope--
what's the hope
with CRISPR technology
in the treatment
of autoimmune
diseases,
and is there actually
any scientific evidence
that that could
come about?
>> Yeah, that I
actually don't know.
I will tell you, I was
making the analogy
that the reason they
have this little--
like, the Cas proteins
actually recognize
this little piece
right here
so that they don't go and
cut their own DNA up.
So they don't-- the
bacteria don't really have,
in a sense, an
autoimmune disease
where they'll cut,
'cause the bacteria
has no interest in
cutting itself up.
So it has a mechanism
where the piece
it nicks out
of a virus,
it has to be next to
a specific sequence.
And the most
common one is N,
which just means
any amino acid--
or excuse me, any
nucleic acid, G-G.
And so, it'll search
along here, see A-G-G,
and then clip out this piece
to incorporate into its DNA.
But it doesn't put
N-G-G into its DNA,
because it would go
and cut itself then.
And so, it was more of a--
I haven't read much
about autoimmune diseases
in CRISPR, though.
I mean, autoimmune
diseases are such--
are typically where
you have a component
that's self-recognizing.
So I guess, could you
go and cleave out
that section in
your immune system?
Boy, potentially.
But I'm sorry, I don't
know that much about it.
Sounds like you
have a project now.
(chuckling)
Other questions?
Oh.
>> I was wondering-- it's
kind of a two-parter--
is CRISPR interference
preferred in any way over
using an siRNA or microRNAs,
or is that like a
quality check step
before proceeding on, like
seeing a downregulation
in the protein before
actually trying to modify it?
>> Yeah, I think,
that's pretty specific.
I know RNA
interference is--
I would think that this
could have a higher affinity
for a piece of DNA, 'cause
you know that any bacteria--
or excuse me,
any protein,
you can do kinetic
studies on it
where you modify it
and see how strongly
it bonds to a
segment of DNA.
So I would guess that CRISPR
would give you kind of a control
that just throwing another
piece of RNA in it
to interfere wouldn't
provide, right?
So I would think that this would
be a preferred mechanism.
But I know there are definitely
therapeutic treatments
where they just are putting
RNA to interfere with proteins
being created, so I would
think this would be preferred.
Does that answer
your question?
Was there another
part of it?
>> Any other questions?
>> Oh, Taylor.
>> Do you know if there's been
any research in using CRISPR
to develop vaccines or
anything like that,
as far as altering the immune
system to be preventative
in seeking out certain viruses
that we can't right now?
>> (forceful exhale)
I don't.
I will say that I did
read a few articles
where viruses will put latent
bacteria in our bodies, right?
They will insert-- and
what they have done is,
with CRISPR, you can identify
really low concentrations
of things.
So I know from a
diagnostic standpoint,
that's where I think a
lot of this future is,
is in diagnostics.
So like, if you had a virus
still in your system
but you weren't
showing any symptoms,
could you actually go
and clip that out
or identify that it's
even still there?
But in terms-- the immune
system's pretty complex.
(chuckling)
I'm not an immunologist, so...
nor do I wanna give a talk
on it next year, Tim.
(all laughing)
Anything else?
>> So Bill, there's a
lot of controversy
about where this should
or shouldn't be used.
Where do you think, or where do
scientists in general think,
are the least controversial
uses for CRISPR?
Where will we see this
come along first?
>> Yeah, so some of that
work that I read on--
with exosomes is
I do think that--
so ex-- so cells
apparently put out--
they communicate with
these components
that will have proteins
and DNA in them.
I would think that they could
target specific organs
or disease using this system,
as opposed to like--
which wouldn't be that
controversial, right?
I think-- what I
will tell you,
the more I read
about this,
the more I see it
as a diagnostic tool
as opposed to a tool
that's gonna go in
and modify
people's DNA,
unless it's on germline
cells, truthfully.
And it's because it's
pretty unpredictable
where this Cas
protein will, one,
how long it'll
stay in a system,
and when as long
as it's there,
it's cutting DNA.
And they get
off-site cuts.
It's not as specific as
it's being touted as.
So...
I think that.
I mean, if you had--
it's like any drug.
I think that if
you were desperate,
you would try it, right?
(chuckling)
So I mean-- but I think that
where you do see issues
and people don't like to see
are germline modifications
that result in
specific traits.
But using it
for disease,
I think those are the types
of things you'll see.
All right?
>> Maybe one more question,
if there
is any?
>> There might
be cookies left.
Oh.
(all laughing)
Yeah?
>> So because CRISPR
keeps on cutting DNA,
as you've said, because it's
not exactly well-regulated
at the moment, if it
keeps on cutting DNA
in areas that you
don't want it to,
couldn't that be a
limitation of it,
where it would cause its
own kind of diseases?
>> Absolutely.
So one of the things that
they're doing, though,
is that beautiful video with
it kinda looping around,
is there are some
of those domains
in the Cas protein,
they're modifying,
so they don't recognize
the DNA as well.
So they can lower
the affinity
that that Cas9 system has for
a particular segment of DNA.
So it'll periodically
find its target and cut.
But if they lower
the affinity,
the off-site
cuts don't--
it's sort of a give
and take, right?
You lower the affinity
for the cutting.
You don't cut
as often.
But you don't hit your
target as often, either.
And so, I think a
lot of the research
is going to be around modifying
that so it doesn't hit as much,
because what you don't
want is to fix one problem
and create
another problem.
But that's real.
Oh.
>> I lied.
>> Yeah. (chuckling)
>> Can Cas9 bind
regions of DNA
that are methylated more so
than regions that are not?
>> Oh, I don't
know about that.
I know where you're going
with that, but I'm not sure.
I think-- I have read
that they will cut out
areas of
methylation
so that they don't have the
effects of what that might do,
like turning a gene on
or turning a gene off.
But you can get Cas
systems that recognize
specific methylated
areas, though.
So they're
working on that.
I just don't know
that much about it.
All right.
>> I had a quick question.
When they do-- you were
saying that it's easier
to use probably when
you take the cells
out of the patient
and modify them.
Is that what they're currently
doing with immunotherapy?
Are they using the Cas9
to do that, and how come?
Why would that be easier
to get that in those cells?
>> Well, I think it's
because they don't
have to deliver it
through your system,
like inject it into
your blood or--
so they have it in a
particular container
that they can-- 'cause you
can change the solvents
around cells and make them
take things up easier,
'cause you can put more
extreme conditions on a cell
in a test tube than
you can in your body,
because you
have to live.
And so...
(laughing)
so you don't wanna do
too many extreme things
in a biological
system.
But if they can
pull the cells out
and then put them back
in, like bone marrow
and things
like that,
I have seen that they're
doing some success.
But I don't know if they're
doing it in humans yet.
But you see mouse models
and things like that
where they are,
so, yeah.
>> All right, well,
we wanna respect--
>> Thanks for coming.
>> Everybody's time.
And let's give
Professor Faber
another round
of applause here.
(applause)
