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visit MIT Open Courseware at JOANNE STUBBE: Because we
haven’t gotten that far in class to understand
what this protein is that’s the focus of
the paper, I still think the paper is
straightforward to understand. I’m just going to
put it into context. So I was having trouble
trying to decide what to do. And maybe I shouldn’t
have done this. But the fact is
that this technology we’re going to be
focusing on in a very sort of simple way, CRISPR-Cas,
has taken the world by storm. And that’s the take
home message from this. So you can sort of
get what it does. But really to look
at the details, you have to go in and study it. And every time you pick
up another journal, you look at Google
Journals or something like– there’s another 100, 200,
300 papers published on this. So this is a current technology
that has taken off really since 2012. And so very rarely is technology
successful in that short period of time. And it happens to
have been applied to one of the key enzymes
that people are now focused on in terms of
controlling cholesterol levels, which is what
we’re talking about. So I use it as an
opportunity to just show you what this technology is. Have any of you ever
done this technology? Nobody in the last class had
done the technology either. But my niece is
a sophomore here. She spent a whole EUROP
doing this technology. So this technology has
moved into the lab. My lab hasn’t used it either. So I probably can’t
answer any of the details. But it’s one of
these things that it is extremely complicated. I think I can give you a cartoon
overview of how it works. But if you’re going to use
it, just like every tool, you have to study
it in more detail. OK. So I am going to
ask you questions. And this is going to be
different from the one I did on Thursday, because I
spent too much time talking about this article. And then I’ll come back to– how many of you
read these articles? How many of you didn’t
read these articles? OK. So your class is much
worse than the other one. The other one had
read all the articles. OK. So we won’t have a very
good discussion about this. And I’ll tell you why I
think you should read that, but I’m not going to
focus on that till we end. OK. So the paper we are going
to focus on is this one. And this is the gene
product, the protein that has become a focus of
attention of many people in terms of controlling
cholesterol levels and as an alternative to statins
or maybe better than statins, but we haven’t gotten there yet. OK. So this is still on the
drawing board in there. Many people focused
on clinical trials targeting this
particular protein. And so one of the
questions that this paper focused on and asked– hopefully, you all
have read the paper. It was only three pages, so
it wasn’t very hard to read. Is this protein
important in terms of controlling
cholesterol levels? And they did experiments in
tissue culture and in mice to try to address that
issue using CRISPR-Cas as a way of destroying the gene,
the gene which then destroys the protein. OK. So this other article
sort of gives you an overview of the kinds of
things we need to think about to make the technology better. And when technology
is introduced– just like if you look
at unnatural amino acids that you guys looked at
with the Schultz technology. I mean, first 15 years Peter
was doing that, he collaborated with my lab. We never published
a single paper, OK? Because the technology
was not good. And so now, the technology
is still not good. But it’s getting there,
and it’s improved greatly. So you often see something. It looks, oh my
goodness, you know, this is going to be fantastic. But the devil is
in the details, OK? And that’s one of the take-home
messages from this course anyhow. OK. So what I want to
do is just give you a very brief overview
of what I had hoped to get to by the
end of lecture today and didn’t quite get there. And so we did get to the fact
that we made LDL particles. And LDL is transferred
in the blood and is a major carrier
of cholesterol. So it takes cholesterol
from our diet. And it’s going to deliver it
into different kinds of cells, and it does so. There’s a receptor on
the surface of the cell. And these little things
here, these little flags, are at the receptors
and the receptors. That’s what we’re going
to talk about next lecture is low density
lipoprotein receptor. OK. And this is the basic. Brown and Goldstein figured
out that genetic mutations in this receptor and
other steps associated with getting the receptor to the
surface of the plasma membrane are responsible for children
for the disease familial hypercholesterolemia where kids
die at age 7 heart attacks, because of inability to
control cholesterol levels. OK. So I need to just
sort of briefly walk you through the model,
because that model is related to the effect
of this protein you were reading
about in the paper that you were supposed
to read for today. OK. So this lipoprotein can
bind to the receptor. This is a plasma membrane. You see there are
three receptors. The receptors have to cluster
to be successful at somehow, by mechanisms that are
moderately well-understood, can engulf the LDL particle
and form a little vesicle. And the little vesicle is coated
with a protein called clathrin. OK. And we’ll see over the course
of the rest of the semester this is used over and over
again– so is clathrin– as a way of taking up
nutrients into the cell. So this is a major
mechanism of doing that. And then what happens is
the clathrin is removed. Biochemically, it’s
removed enzymatically. And what you’re left
with is a vesicle that then fuses with an endosome. And so that’s a little
organelle with lipid membranes that is acidic. And when the LDL protein
gets into the interior of this little vesicle and the
pH is lower than the normal pH, goes from 7 and 1/2
to 5, the LDL receptor dissociates from
the LDL particle. And so then what happens, by
mechanisms that are really incompletely understood,
the receptors can recycle to the surface. OK? And what happens is
that, when some of them recycle to the surface, you’re
left with an LDL particle that fuses with another
organelle called the lysosome. And then this LDL particle
goes into the lysosome. The lysosome is sort
of like a proteasome. It’s a bag of
proteases and lipases. So it just degrades everything
in there– amino acids, fats, everything– allowing you to produce
amino acids and cholesterol, free cholesterol. And then cholesterol in
the liver is often stored. It gets esterified. And it’s stored as
triacylglycerol. Fatty acids esterify
to cholesterols. OK? So the process, of course,
of getting the LDL receptor to the surface is done in the
rough endoplasmic reticulum. Because it’s a
membrane protein, it’s transferred by things called
little coated vesicles. And then somehow these
little coated vesicles deliver the receptor
to the protein. OK? So this is a very
complicated process. And, in fact, mutations that are
responsible for heart attacks occur in every step
in this process. It’s not just the LDL receptor. We’ll see that in
class next time. OK. So the key thing you
need to know for today is that you have LDL receptors
that interact with LDL. And that’s key to taking the
cholesterol into the cell. That’s the take-home message. That’s fairly easy
to understand. OK. So the protein we’re focused
on today is this guy, PCSK9– horrible acronym which
I’ve written down. I can’t even remember it. But it stands for Proprotein
Convertase Subtilisin/Kexin 9. OK. So the important
thing is subtilisin. Has anyone ever
heard of subtilisin? So that’s like [INAUDIBLE]. So it evolved convergently. And so it is a protease that
has a serine, a histidine, and aspartic acid, like you
learned in protein media degradation in the first
part of the module. OK. And this protein
was discovered– we’ll see in a minute– again, because of patients. OK. So the patients presented
themselves in a funny way. That’s how the LDL
receptor was discovered. If you’ve read Brown and
Goldstein’s article, which was one of the things
I asked you to read, you’ve already
gone through that. That was the thing that
got Brown and Goldstein excited about this. What’s going on? Why do these kids have heart
attacks at such an early age? Can we figure out what’s wrong? And can we do
something to fix it? And so, here, what
happens is this protein is made as a proprotein
just like any kind of serine protease. Lots of times you
are pre-proproteins. And they process, they
usually self-process, into an active form. And why do they have that? Why does a protease have
a pre-pro sequence on it? AUDIENCE: So you have, like,
spatial temporal control of the sectors? JOANNE STUBBE: Yeah,
over activities. So you’re controlling
the activity. Because if you
produce a protease, nobody could ever
overproduce proteases. Why? What happens inside the cell? Everything gets degraded. OK. Because proteins
have specificity. But if you overproduce them,
all your proteins have degraded. So it’s not trivial to
overproduce proteases. And so they have a mechanism– hopefully, you
learned about that in introductory biochemistry
course– that makes it inactive till you’re ready to use it. And then it cleaves itself. Something triggers
it, it cleaves itself. And then it’s ready to go. And that’s true here, too. So here you have this
little purple worm that has to auto process
to become active. And in some way, it’s going
to end up extracellularly. And so it’s got to
go through membranes. So it goes through
the Golgi stacks, just like I just showed you
with a cholesterol, the LDL receptor. And it gets extruded
extracellularly. And that’s where it is. It’s out there. OK. It’s processed from the
original version of it. And so the working
hypothesis is– and this was based on a patient. They found a patient where the
LDL levels were elevated, OK. And the child that had this
had early coronary disease. That is heart attacks
at an earlier age. And they studied
this in some detail. And they found out that
what this protein does– I’m not sure we really
understand the details of what the protein does– was that it could bind
to the LDL receptor. OK, so this little orange
thing is what you just saw on the previous slide. And this little
blue thing is LDL. So, now, what happens is instead
of having just LDL, low density lipoprotein and the
receptor, you’ve now got another
protein stuck to this. OK. And so when this
protein is bound, it also undergoes
receptor mediated– they don’t show any steps here. I’m not sure if it’s
been studied in detail. It also undergoes receptor
mediated endocytosis. So it’s taken into the cells. And normally, remember,
with the LDL receptor, the receptor gets recycled. Here, what happens? Something changes
because of this complex. And so now this complex
is in the endosome. But the LDL particle,
which has a cholesterol, doesn’t associate
from the LDL receptor. The receptor doesn’t recycle. But, instead, the whole gemisch,
the protein, the receptor, and the LDL particle,
fuse with the lysosome, which is a bag of proteases. And it’s degraded. So what are the
consequences of that? The consequences of
that are that you lower concentrations
of the LDL receptor on the plasma membrane. OK? And if you lower the
concentrations of the LDL receptor on the plasma membrane,
what happens to the low density lipoprotein concentrations? AUDIENCE: [INAUDIBLE] JOANNE STUBBE:
Yeah, it increases. And so then you’re in trouble. OK? So that’s the model. Again, I haven’t read a
lot of papers on this. The discovery was made
of this of patients that had that phenotype in 2003. But they also found patients
that had a loss of function. And they found out that
some of these patients– they’re different
kinds of patients. They have different phenotypes. But they had a single mutation. And these patients
with single mutation had reduced LDL cholesterol. And they had the same
amounts or elevated amounts of the LDL receptor. And because they
had LDL receptor, they had lower cholesterol
and more LDL receptor to take up the
cholesterol, they had reduction in coronary disease. OK? Everybody get that? Why do we care about that? OK. So can somebody tell
me from the paper what was the take-home
message from the paper? Why do we care about that? What’s unique about
this particular protein protein, PCSK9, compared to
using statins, for example? Did you guys read the paper? OK. So the paper was pretty short. Even if you didn’t
understand all the details, I thought the paper was
pretty easy to understand. So why do we care about? What was the take-home message? Why are we targeting this? AUDIENCE: To change
the expression of proteins that create new– JOANNE STUBBE: PC. AUDIENCE: Yeah. JOANNE STUBBE: Yeah. So but why do we
want to do that? We have statins. Statins, you know, everybody’s
gobbling statins a lot. I mean, you probably know
20 people that take statins. I know many, many people
that take statins. So it’s a wonder
drug in many ways. But when do you
start giving statins? When do people start taking– I’m probably not
allowed to ask that. So you don’t have to answer
if you don’t want to. But are any of you
taking statins? No. OK. But there could be people
that have, you know, high cholesterol. I mean, a lot of it is genetic. I eat McDonald’s
hamburgers all the time. And I eat huge
amounts of ice cream. And I have extremely
low cholesterol levels. OK? And it’s genetic. OK. Other people might not
eat any of that stuff, and they might have
extremely high cholesterol. So when you see people,
maybe your parents, basically, they’re taking this. And it’s after you
have some issue, right? You have coronary heart problem. You have chest pains, whatever. So they start looking for
what could be causing that. And the first
thing they look for is clogging of the arteries. And that’s when they start some
kind of therapy like statins. The beauty of this is,
if this model is correct that I just showed you,
if you could figure out how to remove or greatly
reduce that protein, then that would
automatically, you know, prevent the normal
function of this protein, which is to degrade the LDL
receptor in the lysosome. And I’ll get to that
in the very end. So if you could figure
out how to treat, you could diagnose
the predisposition to having elevated
cholesterol levels and start treating
it much earlier. You have a much higher
propensity for success compared if you take statins
halfway through your life. I mean, there’s really
good epidemiological data that support that. So people are extremely
interested in figuring out– I don’t think we
know the details of the function of what
this protein is– but lowering this protein. Because the consequences
of that are lowering cholesterol in the plasma. OK? So are we all on the same page? Everybody understand that? Because that’s key to
thinking about the paper. OK. So the reason I
picked this is people these people in this
paper wanted to understand is this protein
really important. And so what they
did was they decided they were going to
knock out the gene or do something
to greatly reduce the gene, which then would
reduce the amount of protein, which then would
allow you to analyze the phenotypic consequences. OK? And what was the analysis
they used in this paper? They used two different
kinds of analysis. Well, we’re not in detail. Globally, what did they use? What were their model systems? AUDIENCE: [INAUDIBLE] JOANNE STUBBE: You need to
talk louder because I’m deaf. AUDIENCE: For [INAUDIBLE]
they used a surveyor as– JOANNE STUBBE: Yeah, so that
used a surveyor on what, though? So that’s too detailed. I want a bigger picture. So you’re right. They used surveyor cell assays. That’s more detail
than I want right now. So they looked at it two ways
if you look at the figures. So what were the assays? In the surveyor assay,
what were they assaying? AUDIENCE: The blood samples. JOANNE STUBBE: The
blood samples of mice. So that’s one of the
things from the liver. OK. So they took liver
cells from mice. So they were using
animal models. OK. So one of the questions that,
if you read the paper carefully, you should be asking yourself– and this is always
a question when you’re looking at therapeutics. Is this animal model any good? OK. And then the other way that
they were looking at this was with tissue culture cells. Because, in general, when
you start studying something, you don’t start
on humans, or you don’t start on whole animals. You need to start on
something simpler. And we haven’t
gotten to this yet, but Brown and Goldstein,
if you’ve read the reading, have used fibroblast cells. And they showed fibroblast
cells behave like liver. And it turns out it had
great predictive power. It might not have, but it does. So you need some kind
of a model system. And so they used
both of those systems to try to test the idea
that, if you could get rid of this protein, you
could alter in a way that these patients, these
loss of function patients, behaved in terms of the
levels of cholesterol. So that was it. And so how did they
decide to do this? And so I would
say, in general, we don’t talk about this kind
of stuff very much in 508. But if you’re ever going
to be a biochemist, you can’t do biochemistry
without being able to do gene knockouts
inside the cell. So 25 years ago,
that was tough, OK? In the mid-1980s, you could
first do that well in bacteria. We still do a lot
of that in my lab. It takes four months,
three, four months. It’s not easy. With the older
technology, it works. But it’s a rare
event, and you’ve got to screen through
a lot of things to find the ones
that are interesting. And this technology,
CRISPR-Cas, allows you to do this in a couple of days. It’s revolutionized
what you can do. So you might be studying
something really complicated in the test tube. But the question
is is what you’re studying relevant to what’s
happening in the cell. And so if you’re asking
a chemical question, a mechanistic
question, like how does isopentenyl pyrophosphate
do its chemistry, you don’t need to
do that in a cell. You can do that in a test tube. If you’re asking how
things are regulated, which is what we’re doing
now, you must be in the cell. And the issues
within the cell are that people overproduce stuff. You know, they have
to mess around, so they can see something. And whenever they do that,
they change everything. So the future,
for anybody that’s interested in biochemistry
biology interface, is you’ve got to
be able to do both. And so this technology,
I guarantee, in some form you
will be using if you pursue a career in doing
biochemical and biological studies. OK. So the question
really is we want to do manipulation of a gene. OK? And so people have wanted
to do this forever. So you might want
to delete the gene and see what the phenotypic
consequences are. You can do that. You know, there’s been
technology around. They won the Nobel Prize
for the technology in 1983. But, again, it takes months. And you have to screen
through millions of cells to be able to
figure out which one has your gene deleted or
another gene inserted in place of the gene of
interest where you’ve modified the gene of
interest, which then gives you information about the
function of the protein. And so having technology
that can turn around rapidly is important. And so I’m just going to show
you what the state of the art has been up until two years ago. And, really, they also work
by the same mechanisms. It’s just the CRISPR-Cas,
even though it’s really still early days, works
much more efficiently. OK. So the idea is you have a piece
of DNA that you care about, and you want to cleave it. And all of these cleavages
are double-strand breaks. So double-stranded breaks
are lethal to the cell, so you have to repair them. OK. And you have to have
a way to repair them. And I’ll show you what
those two ways are. You’ve all seen it in some form. But you want to have
cleavage at a specific site. And then when you have
cleavage, the question, if you repair that,
can you delete part of that gene which would
make the entire gene inactive? Or, can you replace,
in this cleavage site, a gene of interest with a
mutation in it, et cetera? OK. You can do many, many, many
genetic engineering projects, which are sort of covered
in review articles. The more creative people
become, the more things you can actually do. OK. So how do you do that? So what they do in the
case of the zinc fingers, does anybody know
what a zinc finger is? Has anybody seen a
zinc finger before? So a zinc finger is a little
small protein, I don’t know, maybe 70, 80 amino
acids that combine zinc and that its sequence
specifically binds DNA. OK. That’s a major way of regulating
transcription inside the cell. OK. And there’s not just
one zinc finger. There are many,
many zinc fingers. OK. So what people have done is
taken these little motifs that combine zinc and
designed these motifs, so they recognize a sequence. So these guys, these
little zinc fingers, now are targeting the DNA
that you want to cleave. So they’re targeting it here,
and the targeting it here. So what that means
is every time you want to do an
experiment like this, you have to make a
little zinc finger. String them together to get
enough binding affinity, so you get specificity. That’s really key. And you could do it. We’re pretty good at
this, but it takes months. And so what they do is, once
they have these binders, then they attach a nuclease. OK. So Fok1 is the nuclease. So that just means you’re
cleaving a phosodiester bond over your nucleic acid. And you cleave on one strand. And on the other strand, you
have a double-standard break. And these enzymes work by
giving you blunt-ended cleavage. There’s no overhangs
in the DNA cleavage. So people have used
this for a long time. In fact, Carl Pabo at MIT, who’s
an X-ray crystallographer that studied regulation by
zinc finger transcription, was one of the people that
founded the companies that got this technology off the ground. But it’s hard. OK. So the second
technology which I think is much more widely used– but I think it’ll be
completely displaced. I might be wrong. You can buy a kit
Golden Gate, TALEN kit. That’s right. You can buy it
from some company. And it’s the same idea. So, I mean, I don’t know
anything about this in detail. But it turns out that these
little proteins, which are 34 amino acids,
you can actually look at a sequence of DNA
and design 34 amino acid repeats in a way that it can
bind to double-stranded DNA. OK. So this is like, so you have
a double-stranded DNA helix. You string a bunch of these
little domains together. And you can actually design
these little domains, the sequence of
these little domains. And it forms a
super helix around the double-stranded helix. So the protein forms a helix
around the nucleic acid helix. And what it does is it targets
the nuclease for cleavage. So it’s the same idea. It’s just the mechanisms
of targeting are different. And so, I mean, they have
structures of these things. It’s sort of really
an interesting problem in molecular recognition if
any of you are interested. But I would say,
if you want to use this to do something
biochemical and biological, you probably want to go
to CRISPR-Cas system now. OK. So both of these are the same. They have a nuclease and
something that targets it to a DNA sequence of interest. And if you’ve read the
paper on the PCSK9, that’s exactly
what they’re doing. They’re targeting a sequence
for double-strand cleavage. OK. So this then brings us
into the CRISPR-Cas system. And I’ve given you a
hand out of this which is, again, a simplification. Now, I think there are
six different moderately well-studied CRISPR-Cas systems. They’re all different. So they all have different
numbers of proteins. Although, the idea of how
they work is pretty similar, I think this has turned out to
be the best behaved in terms of biochemically putting it back
together and having it work. OK. So what do we have here? So, hopefully, you all
know now that what you need for this to work is a Cas9. What’s Cas9? AUDIENCE: The CRISPR
associated with [INAUDIBLE].. JOANNE STUBBE: So a CRISPR– is that what the acronym is? AUDIENCE: …it’s a nuclease. JOANNE STUBBE: Yeah. It’s a nuclease. OK. And what’s special
about this nuclease? AUDIENCE: Sequence-specific. That’s like– JOANNE STUBBE: It has what? AUDIENCE: A guide
RNA that makes it– JOANNE STUBBE: No. So just the nuclease, we’re just
talking about the protein now. We do have to worry
about that, yeah. So if you look at the Cas9
sequence, what do you find out? That’s not in the paper, but– AUDIENCE: So it’s got two
different regions that can bind the two different strands– JOANNE STUBBE: Right AUDIENCE: –and, like,
[INAUDIBLE] in a different [INAUDIBLE]. JOANNE STUBBE: Yeah. So you have two different
nuclease domains. OK. I mean, this is not
necessarily given. One is going to
go to one strand. And one is going to go
to the other strand. OK. And we’ll talk a
little bit about that. And then, as you were saying,
what’s unique about this? In this picture, what’s
wrong with this picture? If you read the original
discoveries in the bacterial system, what’s unusual about
this particular– well, I guess– OK, no. It’s OK. OK. So what do you have here? What is this part? This should be tracr. What’s tracr? AUDIENCE: It’s the
transactivator. JOANNE STUBBE: Yeah, so
it’s transactivating. OK. And then what’s the gRNA? AUDIENCE: The guide. JOANNE STUBBE: So
that’s the guide that is part of this
bigger piece of DNA that we’re going to
look at in a second. OK. So what you need,
although this isn’t what people use now
for the technology, is you need two pieces of RNA. And you need the target
for double-strand cleavage. And you only need
a single nuclease. OK? And the key question is how
do you make them assemble. OK. And how do you make it
as simple as possible, so that you can use
this in bacteria, but also use it in humans which
is what Eric Lander focuses on. So CRISPR, and we’ll look
at this in a [INAUDIBLE],, has this horrible name,
Clustered Regulatory Interspaced Short
Palindrome Repeat. OK? So that’s the name. And so this just
summarizes– and we’re going to come back to this
in a minute– that all three of these methods, the
zinc fingers, the towels, and the Cas9 system,
all do the same thing. They somehow recognize
double-stranded DNA and cleave it, OK? And so they all give you
a break in the DNA, which is lethal if you
don’t figure out how to deal with that break. OK. And there are two ways
to deal with that break. There are two ways of repairing
the break that we’re not going to talk about in
detail, but you probably have heard about somewhere. So what’s the way that they deal
with this double-stranded break in the paper? Did anybody read the
paper carefully enough? And how do they know? So, somehow, you’ve got to put
these things back together. Otherwise, your organism
is completely dead, which is the goal of having this
CRISPR locus for the bacteria. They want to kill
the invading virus. OK. But in this particular paper,
which one of these two methods did they show or did they
propose from the data that they talked about
was involved in repairing the double-stranded break? If you look at this paper, they
describe non-homologous end joining. Because in the end, if
you looked at the paper carefully, when they were
trying to tell whether they successfully got a
double-stranded cleavage, they did a lot of
polymerase chain reactions to figure out whether they
got specific or non-specific cutting. And when they did the
sequencing on this, they could tell, because
of the different mechanisms between these two,
that most of the damage was repaired by
non-homologous end joining. So what happens
with this approach? What happens with
this approach is that the repair is putting
the things back together. When you have blunt ends,
you’ve lost the information from the sequence. And you have a
disconnect and, if you got a couple of cleavage sites
putting them back together, is really tough. And so when you put
them back together, you might have an insertion. You might have a deletion. You might have a frameshift. You get a mess. But then when you look at
the very ends of your gene using the polymerase chain
reaction, what happens is you get a mixture of things. And you can sequence them,
so you can tell something about how the repair happened
at the double-stranded break. So if you have a
double-stranded break, OK, so the question is here
do you have a deletion, so it’s a little bit shorter. Or, do you have an insertion? Or, do you have
an rearrangement? And what you do then is
sequence these things using PCR. And then you can get information
about the mechanism of repair. OK? So the alternative
mechanism– and this is really important
if you want to replace one gene with another
gene, a whole gene, rather than just
removing the gene, which is what happens here. Here, you’ve made a cut in
the middle of this chain. You’ve removed a
few amino acids. Or, you a removed
amino acid, and it’s rearranged a little
bit, so the protein is never going to get formed. Here, what you’re doing with
the homologous repair is you have a template. OK. So if you don’t know anything
about homologous DNA repair, you need to go back and look
into it, your basic textbook, and at least read the
definition of what’s going on. But you have a template. Once you have a template,
you can copy that template and replace one gene
with another gene. So this template becomes
really key in replacing, site specifically, one
gene with another gene and, as a consequence,
one enzyme or protein of interest with another one. OK. So this was taken from an
article by Jay Keasling. And Jay Keasling is interested
in synthetic biology. He’s an Artemisinin in fame. We talked about that in class. That’s the anti-malarial agent. He’s also been a major player
in trying to figure out how to make bacteria use
mevalonic acid pathway, which is what we’re talking about
in class, to make jet fuel. OK. So how do you make
hydrocarbons that are really energy efficient
compared to ethanol or butanol? And so his whole lab is
focused on figuring out how to use CRISPR-Cas
to engineer genes from many different
organisms back into the organism of choice. And this technology,
apparently, allows you to do five or six
genes simultaneously once you figure
out how to do it. And so you can do a
lot of manipulation in a really fast time
compared to the months it used to take before. And so what does this tell us? I mean, I think this is
the most amazing thing. If you read the Eric Lander
historical perspective on the discovery of
CRISPR-Cas, there was a guy in the late
1980s that lived in Spain and did all his research
in a salt marsh. OK. And he got really interested
in these archaebacteria, really weird bacteria. I don’t think they’re
weird, but most people don’t really think
about they have really interesting chemistry. And when he was sequencing
part of this, for some reason, he found palindromic repeats,
many palindromic repeats. And that’s those purple spacers. And he says, well, what that
heck is going on with that? What is this? OK. So he had discovered
this locus in the genome. OK. Now, bioinformatics
over the years, if you read the history of
this, played a huge role. So you go back, and you look
at all of these sequences. You find even an E. coli,
you have these little spacer repeats over and over
again that are palindromic. And so then the
community got really interested in why you would have
a locus that looks like this. And what they found
is, if you start looking at the genes
on either side of it, you found genes
that were conserved, that coded for the Cas9 protein. In this case, S.
pyogenes is the one that was used in this paper,
which is the nuclease. And they also found this
transactivating RNA. And what’s interesting
about the transactivating RNA is it has a sequence
that’s homologous to one of the sequences in the spacer. OK. So they’ve got to be able
to hybridize to each other. OK. So that started. It became very interesting. And the question
was focused on how is this editing
going to happen when you get cleavage of your gene. Or does this act
like, for example, in SI or an SH RNA in
controlling levels of gene expression? And so there were many people
that contributed over the years to figure out how
this locus is used. And that’s what I’m going
to briefly describe. So the idea is the following. And I think that
discovery, in my opinion, is really a seminal
discovery by some guy who was working in a marsh working
on some bizarre archae, made this discovery, followed it
through for the next 15 years, and discovered that bacteria
have adaptive immunity systems. I mean, that’s really
sort of mind-boggling. I remember when this
paper was published, a first paper was
published where they knew this was happening. Somebody in my lab gave
a group meeting on it. And my mouth just
dropped to the floor, because nobody
predicted this at all. This is what I would call
a revolutionary discovery. And what they found
was– and, again, this is the bioinformatics
data analysis now, which we can do better
and better and better. What they discovered– they
were looking for what’s in between these spacers, OK? So what’s in between
these repeats? I’m calling it the wrong thing. These little purple
things are the repeats. Again, they are the
palindromic sequences over and over and over again. What is in between the
repeats of the spacers? OK. Where do the spacers come from? Well, they didn’t know. OK. But when they started
looking at sequences of many of these things,
what they found was they came from phage, viruses. OK. So, here, they have a bacteria,
and they have phage DNA. Because people would sequence
a lot of, at that time, phage DNA. And so what happens
is the virus, or it could be a plasmid born
piece of DNA where information is transferred from one
bacteria to another, they get into the cell. And then they have proteins. And these, again, are Cas genes. And people are
still studying these that take the viral
DNA or the plasmid DNA and cut it into little
pieces and somehow insert it between these repeats. So all of these spacers are
different sequences of DNA that come from the invading
species, the virus, or a piece of plasmid that
you got from another bacteria. And so that became
really exciting. OK? And so then the
question is, how do you take all this information
and convert it into something that can kill the idea– if you have adaptive
immunity, how do you use this information
to kill the virus? OK. So what we now know
happens is that the DNA can be transcribed into RNA. OK? And so you have
this piece of RNA with the repeat and the spacer. And then you can also transcribe
the transactivating RNA. And they form stem
loop structures. That’s what those
little things are. So they have palindromic
sequences– so, you know, the base pair, that’s why they
draw the picture like that– and Cas9. OK. And so what we know now is
that this strand of RNA, this pre-CRISPR RNA can interact
with the transactivating RNA. OK? So they have a way of
hybridizing to each other. And that’s what you see here. So you see this
little purple repeat. And you see the
hybridization there. And these two pieces of
RNA can bind to Cas9. OK. So Cas9 is the nuclease. And in this particular
type of CRISPR, there’s a ribonuclease,
RNase III, which takes off all this stuff. So you only have a single
spacer that’s actually going to be recognized. OK. So you typically could do this. You could do this again
with different pieces of DNA and make many of these
things, OK, and do many cuts. And so that’s why
people in engineering are excited about this. You can do more than
one cut at once. But we’re just going to focus
on a single set of cleavages, double-strand cleavage,
with one spacer. And in this case,
the spacer is brown. OK? So we trim it. OK. And so this is our machine, two
pieces of RNA and a protein. And then it goes searching
for what happens. The virus invades. The virus has this sequence
somewhere in its genome. Somehow, the bacteria knows
the virus has invaded. It makes this machinery. It goes searching
for this sequence. This sequence then
is recognized, because it can hybridize to one
of the two strands of the DNA. OK. And the nuclease then simply
cuts it in two pieces. So the idea is simple. I mean, obviously, this is an
extremely complex process where it’s going to be regulated
at every step along the way. But, somehow, bacteria have
figured out that, you know, if you have a virus that
infects the bacteria, what often happens is the virus
causes cells to lyse. And the bacteria is dead. OK? So that happens. So to save yourself, you
want to get rid of the virus. OK. And so this is a
way that bacteria have evolved to be able to
kill this invading virus that would otherwise kill that, which
is what adaptive immunity is all about. OK. So this is the model. And so then what people
have been focusing on and what was focused on
in this paper is Cas9. OK. So the idea’s easy. It cleaves double-stranded
DNA and gives you blunt ends. Furthermore, it knows where
that occurs relative to a P-A-M site, a PAM activating site. It cuts in a certain region. So they’ve studied all of that. They know where it cuts. I’ll show you that in a minute. And then if you want to target
any gene inside the cell, you now can put in
the right spacer. OK? Then you put the
whole thing together. Now, the key issue is getting
all of this stuff, the protein and the two pieces of RNA. They are going to go in as DNA,
OK, getting them into the cell. And how did they get– because if you can’t
get it into the cell, you can’t do the
double-stranded cleavage. So how did they get
this into the cell then? Anybody notice
that in the paper? AUDIENCE: Adenovirus. JOANNE STUBBE:
Yeah, so adenovirus. So people are trying
to do gene replacements using all kinds of methods. None of this is trivial. In this case, they’re
working on a mouse liver. And adenovirus, I don’t know
very much about adenovirus. But, apparently, it likes
to live in the liver. So that’s one of
the reasons they chose looking at
the mouse liver, but it happens to also be
where all the cholesterol metabolism or the predominant
cholesterol metabolism occurs as well. And so they wanted
to try other things. In the end, you’re
probably never going to be able to use adenovirus. People have been trying to
do that for years for gene replacement without success. So a key issue is going
to be how do you get this into the cell, I mean. And so that’s what
a lot of people are trying to focus on now. But to do this in
tissue culture, you can do it
without any problems. There are ways of
getting it into the cell. OK. So this is what we
were talking about. Once you get the
cleavage, you know, you can repair the
cleavage by this method. If you want to read about
this, you can go read about it. But what this does is gives
you a deletion of your gene. Or, if you have a
template, then you can use this template
to remake a protein with a mutation in
it, for example, or with a tag on the
end, so you can purify it by affinity column,
chromatography. And so then the question
is in almost all cells– and it depends on
the organism– you have both mechanisms of repair. And so one of the
issues is how do you tweak the repair
depending on what function you want to use the
technology for to do this or to do that. And a lot of people
are studying that. That’s one of the
focuses of many labs if you look at what are the
issues, where are we going. OK. So if you look here, we were
just talking about Cas9. So the blue is one nuclease. And the green is
another nuclease. And in this case, this is the
double-stranded DNA target they’re after. And this little piece here, this
TGG, is called the PAM site. And that’s required
for recognition by the Cas9 protein. And do we understand
the basis of that? The answer is yes. I don’t know. Did any of you hear
Jennifer Doudna talk? Yeah. So, I mean, she’s the
one that discovered that. She just published two weeks ago
the structure of this complex. And so, you know, I haven’t
had time to study it. And this is the kind of
thing that, if you really want to use this, you got
to roll up your sleeves and get in there and study it. But what they did
was instead of having the guide RNA and the
transactivating RNA, they put them together. And so that makes the
genetic engineering simpler. But the question is how
do you put them together. OK– not trivial. OK. And the whole Eric
Lander article about the history
of this process, the Doudna, Charpentier
group, figured out how to do this extremely
efficiently for bacteria– doesn’t work in humans. So Lander’s article
is focusing on, you know, humans is
much more important. And so Zhang, who is at MIT,
had figured out another way. You can’t just use these
two little pieces of RNA. You need something much
bigger to have the Cas9 work successfully inside the cell. And what’s the basis of that? I don’t know. But they did a
lot of experiments to figure out how you could get
this to work most efficiently. So these are the two partners. And the question
is what’s going on. And, you, know,
frankly I haven’t even had time to digest. My lab hasn’t used
this technology. I haven’t had time to digest it. But what you see– let me just point
out one other thing. There are two domains. So you have the nuclease domain,
which are not contiguous. And then you have
a helical domain. And Doudna used Cryo-EM, which
we’ve talked about at 25, 30 angstroms resolution–
not particularly good– to show that when you
started with Cas9, but you added the
two pieces of RNA, you got a change
in conformation. They could see that in the
Cryo-EM, because it was huge. OK. And then when you add the
targeting DNA, what happens is the nuclease domains
change tremendously. The conformation of the
protein changes tremendously. Putting everything together, you
have the double-stranded DNA. So here you have the
double-stranded DNA. Here, it’s hybridizing
to the guide RNA. And this is the tracr
transactivating RNA. And the Cas9 simply
surrounds this whole thing. That’s what she talk about
in the lecture this past week or past two weeks. So this was at 30 angstroms. And they had that model. And this was the result of an
atomic resolution structure that just came out a week ago. And it just shows
you, I’ve told you, you know, you have to
separate the strands. I’ve given you a
cartoon of that. And you need to stare
at this a long time. But green is one nuclease. Blue is another nuclease. One can see that the blue
and the purple are the DNA. And you can see the
two strands separating, because they need
hybridize to two different parts of the
guide and the tracr RNA, which is in orange. And so what they’re
doing is looking at a model for how this works. OK. So the key issues,
all of which were covered in this paper in
some form, are shown here. And these are the key issues
that everybody is facing. And I’m already over. But the delivery method into the
cell, OK, we talked about that. They use adenovirus
in this paper. So I would suggest you go back
and you look at what they did. OK. Off target effects,
did they look at that? Does anybody know? Did they look at that in
this particular paper? Yeah, they did. And so how did they figure out
what’s going to be off target? How did they choose
what to look for? I mean, you know, you got a
billion base pairs, right? So how did they tell
what to look for? One of the first things
they did is what? How did they target the PCSK9? How did they figure
out what to target? Can anybody tell me that? All right, nobody
can tell me that? I guarantee you’re going to
have this on the first exam. You’re going to have something
on this in the first exam. We’ll see if you go
back and you read this. So the whole paper, really the
whole first couple figures, is focused on how do you decide
what to target in the PCSK9. And they looked of exon 1. And they looked at exon 2. And then they did experiments
to try to look at that. That’s how they got
this idea about what the mechanism of repair was. And the sequence they
targeted, they then look for other sequences that
were three or four base pairs different. And then they also did PCR
reactions on all those genes to see if you got
cleavage or not. So if this is ever going
to be used technologically in humans, which is the goal
of this paper– you know, we’re very far
removed from that. We have a lot of
ethical questions. The bottom line is
you need to remove all the off-target sites. You need to control, as
we’ve already talked about, the two methods of repair of
the double-stranded breaks. And I think now that
we have structure, we ought to be able
to even better design these three pieces to make
more efficient chemistry of cleavage. I mean, it’s amazing
how efficient this was. So they did the whole
thing in four or five days. I mean, that was
really quite amazing. And so what I suggest you do
now in the rest of the paper is, I think, straightforward. It just tests this
model by looking for what happens to low density
lipoprotein cholesterol. What happens to the receptor? What happens to
cholesterol levels? What happens to
triacylglycerol levels? And does it conform to
the model that people have for the function of
this protein in controlling cholesterol levels? So what I would suggest
you do is you go back now. Hopefully, you’re now interested
in this a little more. And go back and read this. And if anybody
has any questions, they can come back
and talk to me.

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