ADAM MARTIN: All right, so we're
going to switch gears again today, and we're going to move
off of kind of pure genetics and start to talk about
molecular genetics. And I want to start with
the concept of-- let's say you want to identify
a piece of DNA, purify it, and propagate
it so that you have it for future use. And so the process of doing
this is known as cloning. And it's the process
of, if you will, purifying and propagating a
piece of DNA in an organism. So sort of the goal
for this lecture is for you to know how if
you wanted to, let's say, identify a piece of DNA-- maybe you're interested
in the piece of DNA that contains a given gene. How would you go about getting
that DNA in a state that can be propagated sort of on and on? And how can you identify
the piece of DNA you want? And so one tool that
we're going to use is we're going to use an
organism bacteria as a tool. So I'll draw my sample
bacteria cell here. Could be something like E.
coli, just some bacterium. And you'll recall, when I talked
about cells a few weeks ago, bacteria and
prokaryotic cells have a chromosome called the
nucleoid that's present inside. So that's the
bacterial chromosome. But bacteria can also have
extra chromosomal pieces of DNA that are called plasmids that
exist in their cytoplasm. These are plasmids. And these extra
chromosomal pieces of DNA replicate independently
of the chromosome. And they can persist
in the bacterial cell and be passed on
to the daughters of this bacterial cell as
the bacterial cells divide. So if we focus on an individual
plasmid, what a plasmid would look like, often they have
a cassette or a gene that confers antibiotic resistance. And that's often the reason that
these bacteria are harboring these plasmids, because it
gives them a sort of advantage if they're exposed to
a certain antibiotic from a predator organism. So this would confer
antibiotic resistance. One common example is
ampicillin resistance, which I'll abbreviate just
amp with an R next to it. But these plasmids, for them to
propagate from bacterial cell to bacterial cell,
they also need to be able to
undergo replication. So they also have what is known
as an origin of replication, which is often abbreviated
ori, which basically allows this plasmid to be replicated
such that copies of the plasma are passed on to the subsequent
generation of bacteria. And we can take advantage
of this plasmid system in bacteria, because we
could take, let's say, a piece of foreign DNA-- and this foreign DNA could
be of eukaryotic origin. We could take a piece
of eukaryotic DNA and insert it inside
of this plasmid. And we can basically use the
plasma as a sort of platform or a vector to carry
the piece of DNA that we might be
interested in and to use the bacteria to replicate
that DNA and pass it on to its descendants
so that you essentially have a clone of
bacteria, and you have a clone of this DNA in
a given bacterial population. So, again, this would
have an origin and maybe an ampicillin resistance to it. So how would you
determine whether or not bacteria have a plasmid in it? Can you think of
an experiment you could do to determine whether
the bacteria has this plasmid? Stephen? AUDIENCE: You could
add ampicillin, and it'll survive
with the plasmid. ADAM MARTIN: Exactly. So what Stephen
suggested is that if he wanted to know whether
or not this bacteria had the plasmid in it, he would
add ampicillin to the media. And if the bacteria
doesn't have the plasmid, it won't be able to grow. But if it has the plasma,
it encodes this gene that confers
ampicillin resistance, and it will thus
be able to grow. So that's exactly right. So you're able to select for
bacteria with a given plasmid by simply growing it on media
that contains an antibiotic. I now want to go through steps
in cloning a piece of DNA. And we'll go through it sort
of as a series of ordered steps so you can see how
the process works. I'm going to start with
a step to cut the DNA. After cutting the DNA, we'll
then mix pieces together. Once we mix the
pieces together, we'll do something known
as a ligation, and I'll explain that
to you in just a minute. And then, finally,
we'll end with selecting the plasmids that have the piece
of foreign DNA that we want. And this is known
as recombinant DNA, because you've recombined
a piece of DNA from one organism-- it could be
a eukaryotic organism, like humans-- with a piece of DNA that's
from a prokaryotic cell, a bacterium. So we then have some sort
of selection process. So we're going to go
through this step-by-step. And we're going to start
with cutting DNA, OK? So, cutting DNA. And it turns out,
we've talked about-- what type of enzyme do
you think would cut DNA? Just generally, not as specific
as what's up on the slide. What type of enzyme
would cleave DNA? Think about how
enzymes are named. AUDIENCE: DNase? ADAM MARTIN: Yeah,
so Stephen suggested DNase, which is a really
good suggestion, right? So an enzyme that will
cut DNA would be a DNase. Another word for
that is a nuclease. It's some type of nuclease. And the type of nuclease
we're going to talk about is going to be an endonuclease. We talked about
exonucleases, which chew DNA from the ends in the
context of DNA replication. But what an
endonuclease is, is it's a nuclease that's going to
recognize a fragment of DNA and cleave it in the middle. So it doesn't require an end. It's going to chop it
right in the middle. And there's a type
of endonuclease, and these are called
restriction endonucleases. They are nucleases that
our natively present in a lot of different bacteria. And these restriction
endonucleases essentially look through the DNA
sequence, and they recognize a specific
sequence of nucleotides and make a cut right
at that sequence. So I have a few examples
to show you here. The first is this EcoR1
restriction endonuclease. EcoR1's from E. coli, and it
recognizes the sequence GAATTC. So it recognizes
this six-nucleotide . Sequence and it cleaves
the double-stranded DNA on the top strand
between the G and the A and on the bottom strand
between the G and the A, OK? And you can see that the
two cuts are staggered. So when this cut is made, it
leaves the DNA with two ends, and they're sticky, because
there's a five-prime overhang at each end. So each end has
this TTAA sequence. And these nucleotide
bases can base pair with the complementary sequence. So this sequence could
base pair with a sequence that has an end AATT. So these two ends that
I've generated here could stick to each
other, or there could be other ends that
have the TTAA sequence that could stick to them. So another example is
this Kpn1 endonuclease. And this is from a
different bacterium. But again, it cleaves the
DNA on the two strands. And this time, it
cleaves the top strand farther down the sequence. And that generates what's known
as a three-prime overhang. But again, you have an overhang. So this is what is known
as having a sticky end. Because again, these
nucleotides are available to base pair with
a complementary sequence. The final type of
restriction enzyme that I'll tell you
about is EcoR5, which is a different
enzyme from E. coli. And this generates a
break, but this time, it cuts at the same position
on both DNA strands. And that generates an end
that's known as a blunt end, because there's no
overhang, and there are no nucleotides that
would sort of basically recognize a complementary sort
of end like the sticky ends do, OK? So these are several of
many, many different types of restriction
endonucleases that are present in a wide
range of bacteria. So then, now that
you have a tool that allows you to cut DNA,
you could then cut DNA from two different sources. And I've outlined that here. The vector is what the plasmid
DNA is commonly referred to. So we commonly refer to
this prokaryotic part of the plasmid, the
vector DNA, and the part that we're trying to insert
that's the foreign DNA, the insert. That's just kind of
the lingo in the field. So here, I have a plasmid. It looks like a plasmid. It has an origin of replication. It has ampicillin resistance. And it has this EcoR1 site,
which just means that this DNA sequence has a GAATTC, OK? So it's something that will be
recognized by this restriction endonuclease. And then if you cut
this enzyme with EcoR1, you start with the
linear piece, right? So I, at 6:00 AM, started
engineering this here. So let's say I have
my plasmid DNA, and I cut it at the EcoR1 site. Then I cleave it. If you cleave a circle, now you
have a linear piece of DNA, OK? But it has sticky ends, right? And these sticky ends-- so pretend that this is
a foreign piece of DNA. This is my eukaryotic DNA. Let's pretend it carries
the gene elastin. And this has ends, too. And if they're EcoR1
ends, then they will be able to stick to the
sticky ends of the plasmid. And if you just
get one sticking, now you have this
piece of DNA which is two different
fragments in tandem. But it's going to
be moving around in space in the cytoplasm. And at some point, it might
be recognized and stick to the other EcoR1 end. And then you have now a
circular piece of DNA again, but now your
circular piece of DNA has this piece of
foreign DNA that's present inside the vector,
which is the sort of poster tape here. So I just wanted
to show you that. So you can kind of-- when you're doing
molecular biology, you have to imagine
the sort of end stick sticking to each
other and how they're going to sort of wrap around and
connect for the final product. OK, so let's say you get
DNA, and your DNA could be eukaryotic DNA from,
let's say, humans or flies or whatever your
favorite eukaryote is. And in the genome
of that organism, there will be many
restriction sites. But if you chop it
up, you will get various fragments that
have sticky ends for EcoR1 on both sides. And then if you mixed the
vector and the insert together, you have some probability
of getting that insert to be incorporated
into the vector. And then once this is
present and ligated together, you can then put
this into bacteria, and it will be replicated. So we focused on cutting, but
if you mix these together, like I showed you
at the tape, you're going to have sticky
ends that come together and stick together. And you'll eventually
get a situation where you have your insert-- so you have your
insert here that might have your gene of interest
and your vector DNA here. But when these things
initially stick together, you don't have a single
molecule where everything is covalently attached, right? You just have these base pair
interactions between the two overhangs as they
stick to each other through base pair interactions. So if we think about
what's going on right here, you have, initially, if
we're thinking about EcoR1, a sequence that is-- oh, sorry, this
should be a C. This is the nucleotide sequence, but
when it's sticking together, the top strand will have a
free five-prime phosphate on this adenosine,
and there will be no covalent bond between this
adenosine and this guanosine. So that's what the top
strand would look like. The bottom strand
would look like this, where there are covalent
bonds along the DNA backbone. Sorry. Had a mutation there. And this is incorporated
in a broader sequence. And this bottom strand will
have a free five-prime phosphate here and a free
three-prime hydroxyl here, but there's no
covalent bond here. There's no covalent
bond here, right? So, at this stage, you don't
have a single piece of DNA. You have two pieces of DNA that
are interacting with each other through base pair interactions. So, eventually, you want it
to be a single piece of DNA. And so you have to perform
a step that is known as-- sorry, my phosphate
got in the way. But you want to perform what's
known as a ligation, where you take something that's
just sticking together through nucleotide
base pair interactions and you add a type
of enzyme, which is called DNA
ligase, to catalyze the formation of covalent
bonds here and here. So DNA ligase is an
enzyme that if you have a three
five-prime phosphate here and a free
three-prime hydroxyl, it'll catalyze the formation
of a phosphodiester bond between these
two bases and the DNA. So this DNA ligase forms
a phosphodiester bond. And you go from having no
bond there to having a bond. So then you eventually
would get this sequence now, where you have covalent bonds
between each of the base pairs. And what you see here is you've
recreated the EcoR1 site. So when you get two
EcoR1 sticky ends sort of recognizing each other
and sticking to each other and then you ligate
them together, you recreate that nuclease site
so that you can cleave it again with the EcoR1 enzyme. So now, moving on. I'll move on right here. So the last step is that
once we have pieces of DNA with this insert-- and let's say we're trying
to find a piece of DNA from a eukaryotic organism. We might start with an animal. We have to extract
its chromosomal DNA, digest it with a
restriction enzyme, and then we digest the vector
with the same restriction enzyme. And then we're going to make-- we're going to randomly
insert these pieces of DNA into different vectors
such that each bacteria who gets one of these
plasmids will be replicating a different
piece of DNA that's of eukaryotic origin. And this is what is
known as a DNA library. So this is making a DNA library. And a DNA library is
essentially a collection of different pieces of DNA
that are from some source, OK? But different bacterial
clones will be replicating a different piece of that DNA. So you can see the
challenge now is to find the needle in
the haystack, right? You're trying to find that
one piece of DNA which is the one you're interested in. And I'll talk about
several strategies that you can use to
find the piece of DNA that you're interested in. I'll focus on selection. But first, I just
want to differentiate between two different
types of ways you could search for a piece of DNA. You could do a screen. And this is similar to what we
talked about on Monday, where you look through a whole
population of individuals, and you look for
a given phenotype. So in the case of
flies, we talked about how Morgan's
lab was looking for differences in eye color. And that was a
screen, because they looked through a
ton of normal flies to find the one they want. Another strategy
would be to do what's called a selection, where you
kill off everything that's not what you want by making
the organism grow in some sort of
selective condition. And then you only
allow the organisms to grow that are the
ones that you want. So this is called a selection. And I'm going to illustrate
several examples of selections, just so that you get an
idea of how this works. So the first example I'll
give is antibiotic resistance. And as Stephen so kindly pointed
out earlier in the lecture, the way we can select for
bacteria that have taken up this plasmid is to
select the bacteria that grow in the presence
of the antibiotic. So let's say you had a
population of bacteria and let's say this
started out being sensitive to an antibiotic. You could transform
them with DNA from a strain that's resistant. And maybe that resistant
strain has a plasmid that has a gene that confers
antibiotic resistance, in which case, if it's taken up by
this sensitive bacteria, if you then grow it on a
plate that has the antibiotic, you might get a colony
or a clone of cells that has taken up
the piece of DNA that you're interested in--
in this case, the piece of DNA that confers
antibiotic resistance. Everyone see how that works? So you're selecting
only the cells to grow here that have taken
up this antibiotic resistance gene. I'm going to use another
example now from yeast, and it involves functional
complementation. And I'm going to
start with something that involves the biosynthesis
of an essential amino acid. And then I'm going to go to a
more interesting case, which is a case that involves an
experiment that involved the identification of the master
regulator of cell division in humans. But we'll start with just
amino acid biosynthesis. And there are mutants of
yeast known as auxotrophs. And these are mutant yeast,
or mutant microorganisms, that fail to produce
an essential nutrient. So an auxotroph is a
mutant that fails to make a nutrient that's essential. So fails to make a nutrient. And the opposite of an auxotroph
is called a prototroph. A prototroph is essentially
a normal-functioning microorganism that's
able to produce all of the essential nutrients
that it needs in order to grow and survive, OK? So this produces all nutrients. And so let's say you had
an auxotroph for leucine. So you had a strain that if you
didn't provide leucine to it, it would fail to grow. So we'll start with
a leucine auxotroph. And let's say you
want to identify the gene that's defective
in this leucine auxotroph. You'd perform a similar
strategy to this one, where you'd have your auxotroph
that you're transforming, so your auxotroph down here. And you would transform
that strain with DNA from what organism? If you're trying to identify
the functional gene, what organism would
you use to produce the DNA you're going to
transform into that organism? AUDIENCE: Prototroph? ADAM MARTIN: Javier's
exactly right. You'd use the prototroph, right? So in this case, you would
use DNA from the prototroph, because the prototroph has a
functional copy of that gene. You know it does,
because it's able to grow without adding leucine. And then you could take the
auxotroph mutants that's transformed with DNA
from the prototroph, and you played it on media. And what should be a
property of the media? Should leucine be
present or absent? Carmen? AUDIENCE: Absent. ADAM MARTIN: It should
be absent, exactly right. So you'd look on
plates that lack leucine or minus for leucine. And you'd select for colonies
that now are all of a sudden able to grow leucine. So you've restored the function
of leucine biosynthesis in this clone, and you've made
it into a prototroph again. OK, this is what's known as
functional complementation, because you're
taking a cell that is defective in some function,
and you're complementing it. You're complementing or
rescuing the phenotype, OK? Now, even as a former
yeast geneticist, I don't find amino acid
biosynthesis and functional complementation in the context
of leucine all that exciting. So I want to present
one last example that involves an experiment that is
going to involve the yeast cell cycle mutants. And I'm going to tell you
about the experiment that led to the cloning of the master
regulator of cell division in humans. And it involves a yeast mutant,
and specifically, a yeast cell cycle mutant. And these yeast
cell cycle mutants are what are known as
conditional mutants. They are isolated as
conditional mutants, meaning that these
mutants are able to grow under certain conditions,
but not others. And specifically, the condition
they used is temperature, so they're
temperature-sensitive mutants. The yeast cells can grow at 25
degrees, but not at 37 degrees. So this is known as a
temperature-sensitive mutant, where you can propagate the
mutant at one temperature, but then you can see if
you raise the temperature, then it stops growing. And so you can see
the mutant phenotype, because normal wild
type functional yeast can grow at
both temperatures. So this is a special
type of mutant. And I'm going to tell you
about an experiment done by Paul Nurse, who's an
excellent yeast geneticist. And what he did was he used
these yeast cell cycle mutants to identify the
human gene for what's now known as cyclin-dependent
kinase, or CDK for short. This is the master
regulator of cell division in organisms ranging from yeast
all the way up to humans, OK? But he used yeast as a model
system to identify this gene. And the process was
he took yeast cells-- and Paul Nurse worked
on fission yeast cells, which are rod-shaped cells. And he identified yeast mutants. Yeast mutants. And he had a mutant in
the CDK gene of yeast. He didn't really
know it at the time. But the yeast CDK mutant-- what he knew was that this
mutant was critically involved in the cell cycle in
numerous types of yeast. So he knew this is
an important gene. And what he wanted
to do was to identify if humans had an
equivalent gene that could function in the same way. So if you just have this CDK
mutant and do nothing to it, it will not grow
at 37 degrees, OK? But what he did was to
take a DNA library-- similar to what I
showed you before, where you just chop up
DNA from an organism. In this case, he's using
a human DNA library. And he used a particular
type of library, but I'm going to skip
over that for now and come back to it later. So he used a human DNA library. That's just a
collection of pieces of DNA from a human source, OK? So he's taking human
DNA, putting it into a yeast plasmid,
and transforming yeast with that human DNA. And he's looking
for a piece of DNA that's able to complement
the CDK mutant, meaning the yeast
cells would then be able to grow at the
non-permissive temperature of 37 degrees. So he then looks for, on
a plate, colonies of yeast that are growing at the
non-permissive temperature of 37 degrees. So if you didn't do
anything with this mutant, if you didn't transform in
the DNA, nothing would grow. But he identified
pieces of human DNA that rescued the phenotype
of this mutant, OK? And so these are yeast that
have the human gene for CDK, and they now grow. And this is a functional
complementation experiment, because you're rescuing
the growth of this yeast now not with a yeast gene,
but with a human gene. And this human CDK gene is so
conserved across eukaryotes that it's able to still
function in a yeast cell, which is pretty amazing. So this just outlines
the experiment here. At 25 degrees,
these yeast mutants can grow and form colonies. And at that temperature,
you can transform the yeast with different
pieces of human DNA. Most of the human DNA is not
going to be what you want. You're looking for the
needle in the haystack. So most of these are not
going to grow at 37 degree. But you're looking for this guy
here that gets the human CDK, and that restores growth
to this mutant strain. So voila, you get a colony
of cells that are growing. And boom, Paul Nurse
wins a Nobel Prize and the rest of the yeast field,
as well, or a number of people who are working on
cell cycle mutants. This is one of the
experiments that led to the 2001 Nobel Prize
for a bunch of yeast cell cycle geneticists. All right, so I've told
you about how to find the needle in the haystack. And this was more common when we
didn't know the genome sequence of an organism. But now I want to tell you how
knowing the genome sequence of an organism would allow
you to replicate and amplify a piece of DNA in vitro. So I'm going to tell you about
an approach known as Polymerase Chain Reaction, or PCR. And what PCR is, is
it's an in vitro method. So it's an in vitro approach
to essentially amplify DNA. And so let's say you
have a piece of DNA-- it could be a piece
of DNA in the genome-- and you know the
sequence of this DNA. And it has base pairs
between the two strands. So, normally, for DNA
replication to occur, what do you need? What needs to happen here? Can a polymerase get in now? No? Why not? Miles? AUDIENCE: The DNA's going to-- because they'll try and
[INAUDIBLE] away [INAUDIBLE] from each other, so you
have to [INAUDIBLE].. ADAM MARTIN: Yeah, you
have to unwind it, right? So you have to
denature it first. So if you do nature it, now you
have two single-stranded pieces of DNA, right? Now what would a polymerase
need to replicate that? Yeah, Jeremy? AUDIENCE: A prime. ADAM MARTIN: A primer. Exactly, right? And if you know
the sequence, you can have a company
synthesize a primer that's the exact sequence here
and base pairs here. And I'll just draw the
five-prime end of the primer right there. And now this primer has a free
three-prime hydroxyl here. And if you added
a polymerase, it would synthesize this
bottom strand here. So this is known as
the forward primer. And on the other
strand, you can design a primer that's complementary
to these bases here. Again, the five-primer
end is out. This would be known
as the reverse primer. And then you could
have the DNA polymerase synthesize the opposite strand. All right, so the step here
will be to first denature. So the first step would
be to melt or denature the DNA, double-stranded DNA. So you denature the
double-stranded DNA. This is commonly done
above 90 degrees Celsius. And then the next
step is once you have these single-stranded
pieces of DNA, you can act you can have a
primer present that anneals to the opposite strands. So you can have
primer annealing. And this is commonly done
between 50 and 60 degrees Celsius. You have to cool it down so that
the primer can now base pair, such that not
everything is denatured. So you have to cool it
down for these primers to recognize their cognate
sequence and base pair with it. And then once you have
the primer annealed to the template, then you
can add DNA polymerase to synthesize a new strand. So DNA polymerize for
new strand synthesis. And this is commonly done at
around 70 degrees Celsius. And then you can repeat this
process over and over again. And at each step, you're going
to double the amount of DNA that you have between
these two primers. So let me just-- this is just
a figure illustrating this. It's on the handout and online. Basically, you have your
original double-stranded piece of DNA. You denature it and allow
the primers to anneal. New strand synthesis. Then you take these new
pieces of double-stranded DNA, denature them. The primers anneal
to those new strands, and now you get new strands. And you just keep doing this
cycle over and over again, and you essentially
amplify the piece of DNA that's between the
two primer sequences. So this is often
used in forensics, because you can have
very little DNA, and just by adding
primers, you can really amplify the number
of pieces of DNA you have between
these two fragments. So you go from having very
little DNA to a lot of DNA. OK, any questions about PCR? I'm going to move on
to something that-- all right. I've really been focused on
discovery up to this point. But I know that a number
of you are engineers, and you probably want
to engineer something. And so I've had to-- I'm going to tell you
about a field that is moving so rapidly, I'm
going to probably have to totally revamp my
lecture for next year, OK? And I'm going to tell
you about genome editing. So the last part of this
story, genome or DNA editing. And I'm going to tell you
about a specific type of system called CRISPR-Cas9, which
has been in the news a lot, and there's a lot of
excitement about this approach in the context of
editing the human genome and possibly curing
genetic diseases. Who here has heard
of CRISPR-Cas9? OK, good. That's good. Our media is doing its job. So who knows what it is? OK, some of us know what it is. I just want to just give you
a very superficial overview of what it is and
why it's important. And I'm going to
keep coming back to it during the
course of the semester, because I think it raises
a lot of ethical questions, and especially in the
context of stem cells. I need you to know the
foundation before we get into the really good stuff. So, let's see. So we're going to
engineer something. So we're going to talk
about repairing DNA. And if we want to
edit the genome, the way this is
most often done is by making a
double-stranded break, OK? So if you make a double-stranded
break in a piece of DNA, it can be repaired
one of two ways. One is by non-homologous
end joining, where the two pieces of DNA
are basically just shoved back together again. And this results,
often, in mutations. So if you're trying
to fix something, unless you're just
trying to break it, that's probably
not what you want. But an alternative
approach to DNA repair that organisms
have is something called homology-directed repair. In this case, you can
break a piece of DNA and add a piece of DNA that
has a different sequence, but with homology near where
the double-stranded break is. And in that case, you can
replace the original sequence with what you
provide as donor DNA. So it gives you an
ability to essentially change the DNA sequence
at a given locus if you're able to cleave
the DNA at a specific locus. So, first, I want to
start with just a thought experiment, right? You're all thinking, OK, we need
to cleave double-stranded DNA. And boy, I just gave you
a perfect tool for that. I gave you all these restriction
endonucleases, right? So what's the
problem with those? Well, let's think
about the human genome. The human genome is
3 billion base pairs. And an EcoR1 site
looks like this-- GAATTC. So it's six nucleotides long. And if you think of
just a random sequence of 3 billion base
pairs, you would get this sequence randomly
one out of every four to the sixth times. So that's going to be
one every 4,096 times. So if you get this in random
DNA 1 every roughly 4,000 times, if you use it to cleave
the human genome, it's going to cleave
hundreds of thousands of places in the human genome. So we need much more
specificity if we want to select,
let's say, a given gene that has a disease-causing
allele and try to fix it. Because if we use a
restriction endonuclease, we just chop up the whole
genome, and that would be bad. So specificity is the
name of the game here. This is not
specific, and we need a tool that's more specific. And that tool is going
to be CRISPR-Cas9. And what CRISPR-Cas9
is, is it's essentially an RNA-guided endonuclease. So it's RNA guided, and
it's an endonuclease. Restriction enzymes, right, they
have nothing to do with RNA. They don't use RNA to recognize
the nucleotide sequence. It's just a protein,
and the protein recognizes the
nucleotide sequence. In CRISPR-Cas9, you have an
endonuclease, which is Cas9. Let's bump this up. So the endonuclease is the-- the Cas9 is the protein. That's the endonuclease. But its selection of a target
depends on an RNA molecule that it's bound to, OK? So the specificity
comes, at least in part, from what's known as a guide
RNA, or single guide RNA. That's what's most often
used in genome editing. So this guide RNA basically
allows this enzyme to find a specific sequence. And the guide RNA
is 20 nucleotides, or looks for homology for
a 20-nucleotide base pair sequence. So you can see, already, we're
doing way better than the six base pair recognition motif. We have 20 nucleotides. And there are other
components of the system which increase the specificity. Then you have your
Cas9 in blue, which is the endonuclease, your
RNA, the guide RNA, in black, and the template
here is in gray. And what you see
is this RNA sort of exhibiting complementarity
to this target sequence. And only if there's
complementarity between the RNA and the target will
this endonuclease get activated and
cleave at this site. So the RNA is sort of
serving like a guide dog for this endonuclease to
guide it to a certain location to cleave. So the idea, then, is if you
want to edit the genome-- and why people are
so excited about this these days is you now
have a system that might allow you to generate a
double-stranded break in one specific place in the genome. And if you can do that, then
if you provide donor DNA that maybe has a different sequence--
if you consider a disease allele, right? Let's say you know
there's a gene that when there's a certain allele causes
an inherited form of a disease. You could then take donor DNA
from an unaffected individual and take cells from
the affected individual and cut the locus
that's problematic and get a repair of the
defective allele using a normal allele of the gene. And that would
essentially rescue the function of that gene
if it were then reintroduced into the patient, OK? Do you see sort of
roughly how this works? So this is a very sort
of broad and general sort of conceptual framework
for how this might happen. Let's say you have an individual
with a blood disorder-- let's say sickle cell
anemia or beta thalassemia. Those are inherited
blood disorders, which lead to anemia. You could remove cells,
and what might be the best are the stem cells
from a patient. And you could then take
those stem cells and use CRISPR-Cas9 in vitro
in cell culture to edit that individual's cells
to repair the genetic defect. And you could then
reintroduce those to the patient, where
if they're stem cells, they'd reoccupy
the stem cell niche and produce functional
blood cells that would then essentially cure
the individual of the disease. This is how scientists
are thinking about the use of
the system nowadays. And this hasn't really
been successful yet, but there are several clinical
trials that are currently underway, where
people are trying to show that this can be used
to treat human genetic diseases. So in the next year, you are
going to hear more about this, almost undoubtedly, as we start
to hear the results of some of these patients. There are concerns
about this, as well. I don't want to overblow it. There are certainly concerns. We don't know this
is going to work. I mean, people have been
talking about this type of stuff since I was a
student 20 years ago. But I feel like we're getting--
we're much more advanced now, and the tools are more advanced. And so I feel like we're kind
of getting to the point where there's a much greater chance
that this will be successful these days than it
was 20 years ago. I just want to point
out where this system-- how it was discovered
and where it came from. And I like this as an example. Much like for the fly
genes that defined major signaling pathways,
this is a discovery that came from
fundamental research on, basically, the
ecology of bacteria. So this CRISPR-Cas9
system essentially evolved in bacteria as a form of an
arms race between bacteria and their predators,
bacteriophage, which are viruses
that infect bacteria. So this is an arms
race between bacteria and their vicious
predators, bacteriophage. And what CRISPR is, where
these enzymes and this system evolved from, is this is a form
of an adaptive immune system for bacteria, which is
pretty wild in and of itself. So CRISPR is an adaptive
immune system for bacteria. If you haven't gotten your
flu shot, you should get it. We'll talk about human
immunity later in the semester. But this is where bacterial
immunity kind of-- I'm sneaking it in. So the way this
works in bacteria-- what CRISPR stands
for is Clusters of Regularly Interspaced
Short Palindromic Repeats. So you can see already thank
god they gave it an acronym. Otherwise, it wouldn't be
getting nearly as much buzz, because no one can say that. And so where this
CRISPR came from is on bacterial chromosomes
of many bacteria, there's these clusters of
interspaced short palindromic repeats, and the repeats
are interrupted by spacers. And what researchers
discovered are these spacers have sequence similarity
and identity to sequences that are from bacteriophage. So each of these
colored sequences here has some type
of complementarity to some type of bacteriophage. And so when a phage infects
bacteria, or some bacteria, what happens is that
there's a system to recognize that
foreign genetic element and take a piece of it and
insert it in the genome. And that serves as a
memory for the bacteria to remember that it got infected
by that particular phage. And then, later on,
what the bacteria does is it transcribes
this region and forms these mature what are
known as CRISPR RNAs, where you can see there's some
sequence would recognize a foreign genetic element. So, therefore, in the future,
if this phage came around again, what would happen is
one of these CRISPR RNAs would recognize the
foreign genetic element through base pair
complementarity, and it would know to cut it. And after the
target is cut, it's then degraded by
the bacterial cell. So this is a way for bacteria
to remember what viruses have infected them and to have
a defense mechanism against it. So it's a pretty cool system. You know, what's also
cool about this system is it's an adaptive immune
system, similar to how we sort of recognize foreign pathogens. What's different about
it is this is heritable. It's incorporated
into the genome. And the more phage the
descendants of this bacteria see, the more of
these repeats you see. So this is a heritable immune
system, which, unfortunately, we don't have. So you should still
get your flu shot. We'll talk about
vaccination later on. Have a good few days, and
I will see you on Friday.
