Welcome to CRISPR Explained: gene editing, history, technology,  and applications.
My name is Damon Tighe. I'm Bio-Rad's curriculum training specialist for the western region
of the United States. If you'd like to
learn more about our products and
services you can contact us at explorer@bio-rad.com or follow the link in
the description. Throughout this
presentation we'll discuss a number of
CRISPR applications that can be used as
hooks for your students and serve as
bridges from your existing curriculum
into this exciting technology. We will
also discuss the mechanism of CRISPR-Cas9 and demonstrate a hands-on
modeling of Cas9 function that can be
used in the classroom. We are currently
in the age of genome engineering and
technically as a species we've been
there ever since we started messing
around with breeding things. Our
technologies have grown over time and
CRISPR represents the largest
technological jump to date with its
extreme precision low cost and ease of
use we start to only be bound by our
imagination when it comes to genome
engineering. You've probably seen
engineering process diagrams similar to
these. If you think about the impact
CRISPR has on these processes in the
context of genome engineering it affects
the entire process because for the first
time we can imagine editing with high
precision cheaply and quickly which
allows us to plan solutions we
previously couldn't have. We are also
getting to piggyback on about 4 billion
years of engineering in the form of
evolution. With this immense potential
comes immense responsibility. The stories
that dappled this presentation should be
thought about as engineering processes
first and foremost that happen to use
biological tools. You should think about
how these stories connect to things you
are already doing in the classroom and
how they may offer an avenue to enter
the subject with students. What do you
need to know for the age of genome
engineering? Well a lot of it you're
already covering in your classrooms. The
basics of DNA and RNA, how those lead to
proteins, the structure and function of
those proteins, how cells work, and a
little physiology systems biology
doesn't hurt either. When I walk into
classrooms and give talks on the history
and future of biotech I notice that a
lot of students come out of their normal
stupor
when we hit the de-extinction projects. This and designer babies will likely be
your strongest CRISPR application hooks
for your students. One of the most
notable de-extinction projects in the
United States is an attempt to bring back
the woolly mammoth, an animal that went
extinct about five thousand years ago
and could be helpful in terraforming
Arctic regions into grasslands so they
don't dump methane into the atmosphere
and accelerate global warming. there are
two teams working on this project: Beth
Shapiroa at UC Santa Cruz is interested in
bringing back the whole mammoth, whereas George church's group at Harvard is
interested in transferring 14 genes from
the mammoths into the Asiatic elephant
to cold-adapt it to northern latitudes. The passenger
pigeon may be the first the extinction
project we see work in the United States
and that's mainly because we have some
really good DNA to start with as the
last one literally died in someone's
hands at the Cleveland Zoo on September
1, 1914. The ecological reason someone
would want to bring back this organism
is it was a keystone species of eastern
forests. Flocks were so big they blocked
out the sun and when they scavenged in
the forests they made major disturbances
that many plants evolved with. Bringing
back the passenger pigeon will likely
have to use the band-tailed pigeon as a
surrogate for CRISPR modified embryos
much in the same way the Asiatic
elephant will be used for the mammoth.
CRISPR could be used to bring species
back but it could also be used to drive
some organisms that humans find a threat
to health towards extinction or at least
under strong local control We have
controlled populations of dangerous
vectors such as tsetse flies and
mosquitoes
with a number of techniques that focus
on producing infertile or failed
offspring. These techniques have been
useful but are self-limiting, requiring
constant manufacture and deployment of
engineered breeding stocks. Gene drives
can increase the success by propagating
a lethal gene or other such genetic
cargo into all of the offspring and not
being subject to that roll the dice scene
inheritance. Gene drives work at a
molecular level by propagating genetic
cargo to multiple sites in a genome. Thus offspring will be guaranteed to
have the cargo no matter which
chromosome is passed on. Instead of
trying to wipe out the vector, gene
drives could also be used to make
species that act as reservoirs for
disease resistant to such diseases.
Borrelia bacteria the cause of agent of
tick-borne lyme disease is present in at
least 43 states and leads to 300,000 new
cases a year. A group at MIT is looking
to use gene drives to vaccinate
white-footed mice. These rodents are the
primary reservoir of the bacteria and
small success has been seen with hand
vaccinate mice but it's not scalable. A
gene drive system that allows the
propagation of antibodies against
Borrelia could allow for a dramatic
reduction in the bacteria after just a
number of breeding seasons. But what do
humans really want to modify? You guessed
it - humans. We have been doing selection
of offspring for a long time via made
choice and contemporary techniques that
allow a pregnant woman to screen for
healthy offspring. But maybe you don't
want to just screen against a major
chromosomal abnormality like trisomy 21.
Maybe you want your child not to have
the cystic fibrosis mutation you carry.
Maybe you wish your child had brown eyes
instead of blue. Or maybe you wish your
child had a cognitive advantage over
others. It's a slippery slope.
CRISPR can be performed on somatic cells
and germline cells. In the case of
editing offspring you're not only
affecting your child but all of their
children as well. Within the past few
years we've already tiptoed into the
latest round of eugenics basically
manipulating the gene pool to select for
certain kinds of people. When you hear
the word eugenics who do you usually
think of? If you're in the United States
it's probably Hitler. But who is doing it
right before Hitler? We were. Cold Spring
Harbor Laboratory has some great
resources on this. Two cases I like to
highlight with students are: 1927 Buck
vs. Bell where the Supreme Court ruled
that it's okay to sterilize women
that have given birth to two or more
feeble-minded children, and Skinner
vs. Oklahoma of 1942 where a judge
stops a man from being castrated for his
third felony which was stealing chickens. You have to remember at this point in
time we thought felony behavior was an
inheritable trait and so to make a
better society castrate the criminals. The topic of modifying people is one of
the easiest places for most people to
enter the conversation around genome
engineering because they immediately
feel skin in the game about what we
should be doing with these technologies
so oftentimes this is a great place to
start with students. I often like to step
them through a couple of questions first
starting with them as parents - what would
they choose? Then have them play the role
of the child that was edited  - what does
that feel like? These first two prompts
help engage the students emotionally
before scaling back to have them think
more broadly about what edits a society
may want to allow or not allow. Then have
them zoom out even further and think
about how does this affect the overall
gene pool and how does eugenics come
into play. It will likely be a muddy but
memorable conversation for you and your
students. A lot of times students might
get the impression that these
technologies just showed up out of
nowhere but remember we didn't get here
overnight. It's important to layout
general histories of transformative
technologies for students so that they
realize these arise through an evolving
process of scientific investigation and
technological innovation very similar to
those engineering process diagrams we
started with. In 1978 we had the first
human born through in-vitro
fertilization and this is big because
it's removing the eggs and sperm,
combining them externally, and implanting
them back into the woman which allows
for potential manipulation of those
germline cells. In 2012, CRISPR-Cas9
hits the scene, and by 2015 China has
already published a paper on modifying
non-viable human embryos. The watershed moment though is 2018 when
Dr. Jiankui He announces that he has
modified three girls' genomes to make
them hopefully not susceptible to HIV. And when it comes to the history of DNA
editing technologies it too was a slow
progression. In 1968 restriction enzymes
are discovered. They are constrained by
only recognizing a limited number of
locations and being able to cut there. This quickly leads to the first big
think tank on genome editing, the 1975
Asilomar conference on recombinant DNA
which sets ground rules for what is okay
or not okay to clone and other safety
precautions all done with the input of
the public. Recombineering technologies
come online in the 1980s making it
easier to move specific pieces of DNA
into a location where a cut has been
made and shortly thereafter zinc finger
nucleases show up. These are modules of
30 amino acids that interact with
nucleotide triplets. By stringing
together different zinc finger moieties
one can create zinc finger nucleases
that specifically recognize sequences of
DNA and can attach there and cut. These
are expensive and time-intensive. In the
late 2000s TALENs come online. These
transcription activator-like effector
nucleases allow for recognition of
individual nucleotides and cloned
together in series it's possible to
program talents that can recognize
chosen sequences and cut there. They are
unfortunately a pain to build and
delivery isn't easy. Why then is CRISPR so exciting?
It's the low cost, the potential for highly
targeted editing, and high ease of use so
much so that you could do CRISPR in your
classroom. CRISPR does make genome
editing easier but remember it's just a
tool in the engineering toolbox and the
system's you are modifying are
relatively complex. For example, if I want
to modify the citric acid cycle in a
cell to get more fumarate out of the
system I could add in more succinate
dehydrogenase or level down the fumerase.
Remember: it's not just about converting
A to B in biological systems. Its rates
ratios and all the other pathways that
might be stealing the molecule you're
trying to do something with. And just to
put this in context let's zoom out from
this tiny pathway and get a look at the
bigger picture. This is an oversimplified
diagram of some of E. coli's major
pathways with the citric acid cycle
circled. You quickly can see how
interconnected the pathways are and the
modifying one position has ramifications
potentially at a great distance. So at
this point it might be good to pause the
video and think about these questions:
What should be our biggest concerns
about genome engineering, and how can we
prepare students for the decisions we
will make as a society with these
technologies? A good number of people
when ruminating on these questions will
wrestle with some grim prospects and
that is on purpose. Humans have a strong
optimism bias that disables many from
thinking slow and calculatedly
about a phenomenon and so occasionally
peppering in a little "how could this go
wrong" helps some people focus. But too
much can shut some people down so let's
look at some non-doom-and-gloom
scenarios. One of the most exciting
applications of CRISPR is in the field
of CAR-T cell therapy where CRISPR
can be used to focus the power of the
immune system on cancer or another
disease. The way it works is T cells are
collected from a patient and edited with
CRISPR so that the chimeric antigen
receptor on the cell can recognize a
specific antigen on say a cancer cell. If
a bunch of these cells are grown up and
infused back into the patient they can
focus the immune system at fighting that
cancer. Another non-germline CRISPR based
therapy could target patients with
sickle cell and this scenario is part of
a capstone activity in Bio-Rad's
Out of the Blue CRISPR Kit. Monopoetic stem cells could be harvested out of a
patient's bone marrow modified using
CRISPR to replace the mutation causing
sickle cell with the wild-type form, grow
up a titer of these cell
and reimplant them which could skew the
patient's ratio of normal shaped red
blood cells to sickle-shaped ones. If
you're worried about CRISPR modified
foods ending up in stores around you
you're too late.
This pizza has two notable CRISPR
modified foods on it. DuPont in 2012
started producing Choozeit Swift which
was a cheese culture that used CRISPR to
fight phage attack in cheese production. Many people don't like mushrooms that
brown after being cut and so engineers
in 2015
used CRISPR to knock out the polyphenol peroxidase that causes browning in cut
button mushrooms. Since neither of these
are adding things to the genomes of the
foods the FDA doesn't require them to be
labeled. Since students are often very
interested in what they eat I like to
give them a chance to play around with
how they might design crops to better
fit their needs using CRISPR. Take one of
these crops and come up with a
modification that you would like to see.
On the next slide, we'll look at a few of
them and talk about where they are on
their way to market. Apples are, not
surprisingly, being modified so that they
don't brown one cut - if you can do it in
a mushroom you can do it in an apple and
same thing with potatoes, but they're
only a proof-of-concept at this point.
If you've ever seen the chemistry involved
in making decaf coffee you may never
want to drink decaf again so groups have
been working on making a decaf coffee
without all of the chemical processing. I
had broccoli to the list only because so
many students hate broccoli that they
often design it out of existence or add
a ton of sugar to it and it's kind of
fun to see where their brains go.
An important story when it comes to genome
editing and agriculture is the attempt
to make hornless cow. Recombinetics, the
company who is trying to make the
hornless cow, after much work got the
phenotype they were striving for and
submitted it to the FDA. Unfortunately, the company didn't check
their work by genotyping. The FDA found
bits of their cloning vector and an
antibiotic resistance gene left behind
in the genome of the cow and promptly
rejected the hornless cow which was very
very
costly mistake for the company. And that
is why any edit with CRISPR should
always be confirmed by genotyping.
How
does CRISPR-Cas9 work?
CRISPR - clustered regularly interspaced short palindromic repeats - comes in many flavors in the
biological world. At its core it is a
bacterial adaptive immune system.
When invaded by viruses who inject their DNA into bacteria, primary immune system
components like restriction enzymes can
cut up the invader. Some portions of the
invader are then captured and added to
the clustered regularly interspaced
short palindromic repeats in the genome
as a way to save in memory of what that
invader looked like. The bacterial cell
and all of its offspring now can produce
a guide RNA molecule that matches the
sequence of the previous invader.
This guide RNA couples with a nucleus
known as Cas9 and if that same virus
attempts to infect the cell the guide
RNA can recognize it and Cas9 can
quickly cut it, destroying the virus's
ability to replicate.
Biotechnologists quickly saw the power in the system because if you could program just a 20
nucleotide sequence as part of that
guide RNA you could send a restriction
enzyme to any location you liked within
the genome. And if you can cut, you can
add to or delete from the genome. The
structure of CRISPR casts 9 system is
relatively simple and demonstrated in
this diagram. Cas enzymes are class of
cutting enzymes involved with CRISPR. Cas9 is the one you will most
frequently hear about in relation to
CRISPR technology right now. The single
guide RNA is a chunk of RNA with
structure that allows it to bind with
Cas9 and contains a 20 base pair
sequence that gives the system its high
specificity by binding to a
complimentary 20 base pair sequence in
the target DNA. the PAM, or protospacer adjacent motif, is a 3-nucleotide
sequence that is just upstream of the 20
base pair target DNA. The protospacer can
be any base followed by GG. In order to
visualize how the CRISPR-Cas9 system
works, the Out of the Blue CRISPR Kit has a
hands-on modeling activity.
You'll be working with three pieces: a strip of
DNA, a single guide RNA with it's 20 base
pair target sequence, the Cas9 enzyme
including its guide RNA binding area, a
PAM sequence recognizer, and nuclease
activity symbolized by scissors.
The first thing it takes place is the single
guide RNA binds to Cas9.
Next the Cas9 and single guide RNA complex loads a
strand of DNA and begins probing it for
the 20 base pair sequence and a PAM
sequence highlighted in green.
Not that the PAM sequence is 3 bases long and
some PAM sequences may use the same base
base of a PAM sequence right next to them.
The DNA strand moves through the complex until the 20 base pair sequence
matches a complementary sequence in the target DNA and the PAM sequence matches.
At this point the nuclease engages and causes a
double-stranded break
CRISPR provides the cut but other
techniques are needed to complete the editing.
Organisms do not like to have
double-stranded breaks in their genomes
and will work quickly to repair them. A
double-stranded break in a bacterial
genome for too long is usually lethal.
Homology directed repair often called
HDR can take place if that cell finds
fragments of DNA that looks similar to
the area around the break and it will
stitch copies of that into place to
resolve a break.
This is by far the most
useful repair mechanism because it gives
the biotechnologists the power to add
things to the genome. The other repair
mechanism is known as non-homologous end
joining (NHEJ) and as a sloppy stitch together
of the double-stranded break that can
result in random small deletions or insertions.
In common speak people will
often refer to this entire process of
cutting and repairing as "CRISPR."
After
students have had the chance to do the
paper model of how CRISPR works it's
worthwhile to dig into the mathematical
modeling how accurate CRISPR is. The Out of the Blue Student Guide can scaffold
them through this exploration and is
really important because students get to
refresh some math skills and use it to
learn how mathematical modeling allows
us to predict some of the complex
behaviors seen with CRISPR.
If you were interested in editing the human genome
which is about three billion, two hundred
and thirty-four million, eight hundred
and thirty thousand bases long and
wanted to figure out what the chances
are of scene sequences of various sizes
I would start by using the table
provided. If we are interested in just
seeing how frequently a single
nucleotide showed up randomly in the
genome we first need to calculate its
probability. Since there are four
different nucleotides, A, C, T, and G, the
probability of seeing A is one in four.
If we then multiply the human genome by
this probability calculation
we are given how frequently at random this
sequence should occur which is
eight hundred and eight million, seven hundred
and seven thousand, five hundred times.
If I was interested in the sequence A-C
I would multiply that 1/4 probability by
another 1/4 probability. I can thus see
that raising 1/4 to the power of the
number of bases in my sequence and
multiplying that by the size of the
human genome tells me how many times I
can expect to see this sequence in a
perfectly random genome.
Since CRISPR uses a 20 base pair
sequence to target the genome we can run
our calculation, the one-fourth
probability raised to the power of 20,
and what we see quickly is that, at 20
bases, the probability of finding that
sequence in a genome the size of a human
is less than 1 which means this is
really accurate in a perfectly random genome.
However genomes aren't perfectly random
so we have to take our results with a
grain of salt.
Off-target cutting is one of the biggest
concerns with CRISPR. How might this
happen and how might it be stopped? These are good questions to have students
think over after the mathematical
modeling activity and to discuss.
Off-target cutting could take place because
you may be cutting a sequence that
occurs more than once in the genome due
to the non-random construction of the genome.
More complex mathematical models
that are based on the sequence of the
human genome could provide better
insight to designing guide RNAs that
land in only one location.
One of
CRISPR's most important uses though will
be one that doesn't get covered much by
most sources as it isn't very sexy but
it is the cornerstone of how we
understand organisms in the molecular era.
That is using CRISPR to disrupt
genes so that we can understand their function.
A stellar example of this was
done recently at UC Berkeley where they
disrupted developmental genes in
butterflies which drastically caused
changes in their wing patterns. Previously this sort of work would have
taken years but CRISPR made it easy to
happen in a very short period of time.
This is really important for the human
genome because we still don't know what
a vast majority of our genes do.
With the Out of the Blue CRISPR Kit from Bio-Rad,
you and your students will
be able to use CRISPR in the classroom
to disrupt a gene that can be monitored
with a color change from blue to white.
How do you know for sure that you
disrupted the gene in the way you intended it?
We don't want any hornless
cow situations.
Genotype it with Bio-Rad's Out of the Blue Genotyping Extension kit.
Now that we've taken care of the hard part, all you have to do is
think about where does CRISPR-Cas gene editing fit into your curriculum.
