ANNOUNCER: This
is the production
of Cornell University.
BUD JERMY: Good evening.
And welcome to the
last summer session
lecture, regular lecture.
Tomorrow night, we have a bonus.
Joyce Carol Oates will be
here and at the same time.
And I would invite
you back for that.
Please silence all
electronic devices.
And I want to also thank
Kathryn Boor, the Dean
of the College of Agriculture
and Life Sciences,
for the use of the hall.
She's been very generous
with us this year.
My name is Bud Jermy, and I'm
from the School of Continuing
Education and Summer Sessions.
And we're glad to have you here.
Ailong Ke is a professor
in Cornell's Department
of Molecular Biology
and Genetics.
He is a member of
the graduate field
of biochemistry, molecular
and cell biology,
the graduate field of physics,
and the graduate field
of chemistry and
chemical biology.
His research focuses
on CRISPR interference
and RNA-guided defense
mechanism in bacteria
and prokaryotic microorganisms.
And his work has been published
in Nature, Science, and Cell.
In 2018, he was the
recipient of the RNA Society
of Mid-Career Award, and also
the inaugural Provost Research
Innovation Award in the
Life Sciences at Cornell.
Ailong has an undergraduate
degree in biology
from the University of Science
and Technology of China
and the PhD in biophysics from
the Johns Hopkins University
School of Medicine.
Before coming to Cornell, he
was a postdoctoral researcher
with Jennifer Doudna at the
University of California,
Berkeley.
Ailong is a leader in
developing a new type
of gene-editing CRISPR system
that he and his colleagues
have used in human cells
for the first time.
This method is a major
advance in the field,
and that is what he will be
telling us about tonight.
CRISPR gene editing moves
out of the laboratory
and into human testing.
[APPLAUSE]
AILONG KE: Thank you, Bud.
So I was told this is going to
be a diverse set of audience.
And so I really
broadened my talk.
And so my own research is
going to be a fraction of it.
And so I want to touch base
more on basically putting CRISPR
into the general
picture and giving you
an overview of where
the industry is
and ethical issues
related to editing.
Maybe some of you are more
interested in hearing that.
And so without
further ado, I guess,
I'll move on to the intro.
And so this is my
attempt to make it
to the general audience.
I might have gone too far.
So if I did that, excuse me.
So I'm a sci-fi fan.
So I started--
I want to start with this movie
by Steven Spielberg, basically
a adaptation of Joyce Wells'
novel The War of the Worlds.
So, the storyline was
that the aliens in one day
basically started their
pre-meditated invasion
to Earth.
They were beamed
into those tripods,
which were hidden on Earth.
And then they fired
these machines up.
And this is when they
make that loud horn.
And within 10
seconds, they start
to fire their primary weapon and
start to basically zap humans
to vaporize humans.
And it was a pretty
desperate situation.
They were grazing
on Earth, picking up
human beings as candies and
converting us to fertilizers.
And no human weapons were
effective against them.
So it was really a hell
kind of a situation.
And so what I liked the
most was the abrupt ending
at the end when just all in
a sudden, they dropped dead.
And so they seemed to have died
from diarrhea or other kind
of diseases.
And so that's the
interesting part.
And so they said
that from the moment
they invade, these
invaders arrive,
start to breathe our air, eat
and drink, they're doomed.
So destroyed by
the tiny creatures
on Earth, which as a biologist,
I assume were the microbes.
And so what I'm
trying to allude to is
why do we earn our
right to survive?
So I want to basically
point out that word.
We have immunity.
So over the billions of years
at the expense of billions
of lives, we
developed our immunity
and our right basically to
survive among this planet's
infinite organisms.
So here, "we" in the movie
refers to human beings.
And so basically all
vertebrates share the same type
of immunity.
So this refers to a subset
of white blood cells, the B
cells and T cells.
And they have an
island in their genome
that seems to evolve
really fast, give rise
to all kinds of diversity.
And that gives rise to
different kinds of proteins.
So in the B cells,
these are the antibodies
displayed or secreted
into the environment.
And then for B cells, these are
the receptors on the surface.
And the B cells were
able to use antibodies
to neutralize the pathogens,
and the bacterias, the viruses.
And the T cells were able
to basically kill all
the barren cells in our body.
And we really benefited
from these cells,
and not only to await the
war against the aliens.
But a rough understanding
of the system
already give us the
technology, the vaccination.
So we start to gain
memories against pathogens
that we have never seen before.
And the hardest therapy
in town against cancer
is this immunotherapy.
So essentially
what I want to say
is that immunity system is
really, really important
for multicellular organisms.
And it turns out that
"we" not only refers
to these kind of organisms.
In microbes-- so these
microbes, the prokaryotes,
also rely on adaptive
immunity system to survive.
This is because they also
face a very tough environment.
So if you just take
a drop of water
from sea or from
Beebe Lake and you
stain for nuclear
acids, what you will see
is that basically DNA is
from eukaryotes, bacterias.
And then in the background,
you see those tiny dots
and they come from
viruses against bacteria.
So on average,
each given bacteria
is surrounded by viruses
in a ratio of 10 to 1.
And so it's a very tough
environment for the bacteria
to survive.
And so here's a picture
of the bacteria surrounded
by a swarm of bacteriophages
waiting to gain access
into the resources inside
and basically multiply
into more viral particles.
And so here are two bacterial
cells kissing each other.
A simple exchange
of genetic material
could lead to a transmission
of a mobile genetic element
or a prophage that cause
death in the recipient cells.
And so over the
years, that bacteria
also evolved a adaptive
immunity system.
And this turns out to be
not a protein-based immunity
system, but a nuclear
acid-based immunity system.
So that's the thing.
A long twist, but this is
where I'm trying to get at.
This involves a CRISPR locus
constant repeats in their space
by variable spacers.
And there's a close
CRISPR-associated operon
involving proteins that
contain activities, nucleuses,
RNA-binding proteins,
et cetera, et cetera.
So when I started my independent
career here at Cornell,
that was 2005.
And the name CRISPR was
never mentioned in--
well, at least I didn't hear
about it in the literature.
And it was one day my colleague
Matt DeLisa knocked on the door
in 2008.
So he was trying to engineer
a metabolic pathway.
And his genes kept
being silenced
by some mysterious
bacteria systems.
And he was diligent enough to
have done a genetic screening
and stumbled onto this
CRISPR-Cas systems.
And he did some
literature search
and found some very
interesting papers.
And so he knocked
on my door and said,
maybe we should collaborate
and study those systems
because you are an RNA expert.
So, the paper he
was referring to
was this paper by Eugene Koonin
So he's an informaticist.
And so based on various
primitive observations that
the variable spacers in the
CRISPR array here seem to match
the sequences in
bacteriophages, the viruses,
and mobile genetic
elements against phages,
and these CRISPR associate
operons contain all sorts
of genes working
on nuclear acids,
and so just based on these
primitive observations,
he came up with the hypothesis
that this entire system is
an RNA-based immunity system in
prokaryotes against basically
foreign genetic acids--
foreign nuclear acids.
And the basic principle
he hypothesized
is similar to the eukaryotic
RNA interference system.
So that was really,
really interesting.
And so if he didn't
point me to this paper,
I would have never read
that journal Biology Direct.
So that was really
serendipitous for me.
And so at a time when we
did literature search,
there were maybe 36 papers
in the entire Pubnet.
So it was really wild
west in this subject.
And so there was just one
paper published in 2007
providing experimental
evidence that CRISPR indeed
is an adaptive
immunity system that
provides protection for
prokaryotes against viruses.
So that was reassuring.
And then around
half a year later,
there was a paper by Erik
Sontheimer and Marraffini,
saying that
interestingly, the CRISPR
were targeting double-stranded
DNA rather than
single-stranded RNA.
So that was
unexpected because we
were looking at RNA
interference, which
target single-stranded RNA.
And it turns out that
implementations already
reported three different
times of CRISPRs.
So it appears that the CRISPR
systems are quite diverse.
So they may have evolved
from different origins.
And so many here probably
knew the name Cas9 here.
At the time, it was
not called Cas9.
It was something called csm1.
And it was one of these CRISPRs.
So, it's a very diverse system.
And somehow it was able to
acquire new DNAs into the array
and acquire new memories.
And based on the analogy
to the RNA interference,
we were expecting that some of
the proteins here in the Cas
operon will form a factor
complex with the guide RNA.
And together, they should
be able to find a target
and somehow destroy it.
The burning question at the time
was that, so if it were RNAI,
we would have understood
it from day one
that basically it involves
[? wasper ?] pairing
between the guide
and the target.
But our target here is
a double-stranded DNA.
How does the RNA find
a matching sequence
that's buried in a duplex DNA?
So that was very
puzzling at the time.
So the other puzzling
question was that basically
if you look at the
guide RNA, so it
has a perfect match
in the foreign DNA,
but also a perfect match
in the CRISPR array.
So how does it distinguish
what's foreign DNA and what
self DNA and prevent
auto-immunity from killing--
cleaning the self DNA and
cause basically death?
So the same question
was answered first.
And so but
informaticists basically
said that the foreign
target is always
flanked by something called
protospacer adjacent motif.
The short sequence identifies--
basically earmarks the target.
And the equivalent position
in the CRISPR array
essentially disallow target
searching in that array.
So then this is self,
and that's foreign.
So the second question
was solved by biochemists.
Essentially they
demonstrated that in order
to find the target,
the effector complex
has to unwind double strand
DNA and basically promote
base pairing between the guide
RNA and the target strand DNA.
And this effectively loops
out the non-target strand DNA.
So, we're forming something
called an R-loop intermediate.
And so that's an essential
process in every CRISPR system
that targets DNA.
And it just turns out that
some time later, people
started to realize that the
nucleuses in the CRISPR systems
almost exclusively target
single-stranded DNA.
So essentially you have
to open the R-loop,
expose DNA in the single-strand
formed, then cleave them.
And this is a quality
control mechanism
to prevent aberrant targeting--
a premature targeting
of a near target.
And things become really
interesting in 2012 when
Jennifer Doudna and Emmanuel
Charpentier published this
paper in Science essentially
reported the biochemical
reconstitution of the
CRISPR/Cas9-based DNA targeting
in test tube.
And then demonstrated
that you can
change the guide
and program the Cas9
to target a different guide.
And essentially you have
a programmable nucleus
that you can use to
target different regions.
And that formed the basis for--
opens the doors to
do gene editing.
And that was a tough
biochemistry experiment.
And they have to
identify that there
are two different non-coding
RNAs that's involved.
And it has to target PAM.
And there are two
single-strand nucleuses.
And when everything
comes together,
you have DNA cleavage.
And then they were
creative enough
to fuse the two guided RNAs
and give you a single guide
RNA that can be efficiently used
in gene-targeting experiments.
And so synthetic biologists
at Boston quickly followed up.
So John and George Church came
up with back-to-back papers
only half a year later and
basically reporting the usage
of CRISPR/Cas9 in human cells.
And did they demonstrated
that they all come from
CRISPR/Cas9-guided targeting
human cells frequently result
in indel formation, so-called
insertion and deletion--
small insertion/deletions as
the result of targeting events.
And they came up
with many concepts
that are still being
discussed in the literature.
They basically formulate
the delivery strategy
of how to multiply this
system, target multiple sites,
how to use just a single
nucleus activity rather
than double nucleus--
double-stranded
nucleus activity,
use two guides to generate
a deletion rather than
a double-strand break, and
the repair pathways involved
in repair the
targeting outcomes,
and the concept of
the off-target events.
And so these are really a
brilliant set of biologists
that really shaped the field.
And from these important
resource, we essentially--
this is the beginning of an era
of efficient genome editing.
So this is not the first
genome-editing tool.
So before that, we
have zinc fingers.
And our colleague at
Cornell, Adam Bogdanov--
I hope I pronounced this right--
so basically pioneered the
usage of TALEN in genome editing
here.
So there are two tools
preceding CRISPR.
So there are certain
traits in CRISPR
that just make it really, really
popular among the researchers.
So this includes the fact
that it's really, really easy
to use.
An average biology lab
can just pick it up.
And nowadays, undergraduates
can be trained
to use it fairly easily.
And this CRISPR/Cas9
tool is super efficient.
And especially the
S. pyogenes Cas9
that's reported in
Jennifer's paper,
this remains the most
effective Cas9 today.
So essentially, they hit
the jackpot from day one.
And this tool is very versatile.
And you can adapt
to different systems
and get different outcomes.
And it really allows
the researcher
to break the barrier of having
to work on model organisms
where genetic tools
are available.
And now they can chase
interesting biological
questions and go to
exotic model organisms
to do genetics and
understand the outcomes.
And there are some
limitations of this tool.
And that becomes obvious
very quickly as well.
And so the Cas9 in
particular is favoring
efficiency over accuracy.
So it's prone to
off-targeting effect.
And when it comes
to therapeutics,
this is a concern.
So you don't want to
edit a size that's
not intended to be edited.
And in order to achieve
editing, as I mentioned,
you have to have a
PAM sequence last
earmarking the target site.
And so the PAM
sequence restriction
here really limits the
targetable sequence space.
So we had-- if you want
to repair some place,
it has to be flanked by a PAM.
And there are also
some other limitations.
So it's hard to
predict which site
can be efficiently edited,
whereas others are not
so efficiently edited.
It seems to be--
there's some randomness there.
And the outcome of the
editing is hard to predict.
And I'll explain
that in details.
I won't go into details about
all the mechanistic work.
But there was a huge line of--
there is huge interest
from the community.
A lot of labs contributed
to important studies
to understand the structure,
the function, the mechanisms,
and how to make good use of
the system, so high resolution
structures,
single-molecule studies
of this dynamic behavior,
and directly evolution
to make it more useful,
et cetera, et cetera.
So there are interesting
studies to understand
the PAM recognition, which
is not depicted here.
But based on that
structure study,
they were able to come up with
rationally-designed new PAM
codes for Cas9 to some extent.
And they were able to come
up with creative basically
ideas or rational designs
to generate high fidelity
versions of Cas9
that's more accurate,
and there's less
off-target effects.
And they were able to come up
with new activities from that.
And so I won't go
into the details.
So I want to reiterate that the
default activity of CRISPR/Cas9
is to cleave and destroy DNA.
And that's the outcome of the
RNA-based immunity system.
And in Cas9 system,
this is the formation
of a double-strand break here.
So the final outcome
is the outcome
of the repair by the host cells.
So in human cells,
the default repair
pathway to deal with a
double-strand break in most
cases is a process
called non-homologous end
joining process.
And so basically a
double-strand break
is a crisis situation
in the cells.
And the cells try to
patch it up by polishing
the ends to a blunt end and then
quickly ligate them together.
So this process will
trigger a lot of error.
And so if the targeted site is
in the protein coding region,
these errors will result
in auto-frame insertions
and deletions and
loss of function
in the targeted protein.
And so in many cases,
the uses of CRISPR/Cas9
is to cause
out-of-frame mutations
in the targeted genes.
So if you want to achieve
very precise editing,
you want to provide a
template and promote
the cells to use the
homology-directory repair
pathway.
So essentially this requires
resection of the DNA,
and invading into
the template DNA,
and copying off the
information from that.
And so that's not the default
repair pathway in the cells.
You really have to
do a lot of work
to essentially coax the
cells to use this pathway.
And so usually it's
not very efficient.
So if you combine all
these things together,
the take-home message is that
essentially, in most cases,
if you do
CRISPR/Cas9-based editing,
the outcome is not
so predictable.
But it's usually a disruption,
a loss of function editing
outcome.
And another drawback
in using this tool
with the idea of
to do therapeutics
is the problem of the
off-target effect.
So I mentioned that Cas9 is not
inherently the most accurate
at editing enzymes.
So it favors efficiency
over accuracy.
So there are some other enzymes
that are inherently more
accurate, but the trade-off is
that they're not as efficient.
And so the off-target
effect basically
refers to the modifications
of unwanted sites.
Usually these are highly
homologous sequences
that are a few base pairs
different from the intended
target.
So, if you have the luxury
to target not just one site
but a stretch of
sites, then you can
use informatics to choose
the site that's quite unique
and avoid the sites that could
trigger an off-target effect.
And so there are
some creative ways
to engineer high
fidelity Cas9s that
solve the problem partially.
And it became obvious
that the longer
you expose human DNA to
Cas9, the more likely
you will get off-target effect.
So really reducing
the contact time
is an important strategy to
avoid off-targeting effect.
So you don't want to
deliver Cas9 permanently.
You want to have a
transient delivery effect.
And if you can avoid delivering
DNA, then delivering mRNA
is better.
Or better yet, if you can
deliver RNA protein complexes,
which will only last a
few hours in the cells,
then the off-target effect
become dramatically reduced.
So there are many
creative approaches
being created to circumvent
this off-target effect.
And yet one approach involves
introducing a shut off switch,
the so-called Anti-CRISPRs
that's naturally occurring
and was used by
the bacteriophages
to inactivate the
CRISPR systems.
And so here the researchers
introduced the Anti-CRISPR
and basically shut off the
Cas9 after a certain exposure
to the human cells, so a
lot of creative research.
So I want to switch
to therapeutics,
I guess, with the idea
that there are limitations
to the CRISPR/Cas9 tool.
It's a very, very powerful tool.
But we understand that it
has off-target possibility.
And the editing outcome is kind
of unpredictable in many cases.
So then with these
understandings,
there are ethical concerns when
you use them in human patients.
So, it's one thing to
use it on somatic cells
without the possibility of
passing the edited cells'
genetic information to
the next generation,
so-called somatic editing.
And so you can do it
either ex vivo or in vivo.
Ex vivo is even safer.
It involves taking the
cells away from the patient,
do the editing experiment,
and then putting it back
to the patient.
So the in vivo editing really
involves really injecting
the editors into the
bloodstream or localized areas
and try to achieve
editing outcome.
This stands in contrast to
so-called the germline editing,
so where you're really editing
an embryo or the gametes.
So here, this is
really, really dangerous
because we're really passing
the genetic information
to the next generation.
If we cannot control
the editing outcome,
we're basically passing
whatever the failed experiments
to the next generation.
And from the patient's benefit
from the species as a whole,
human being species, this is
something we don't want to do
if we cannot do it safely.
And the ethical concerns varies
depending on the situation.
So if is a
life-threatening disease,
then it's easier to gain
ethical approval to carry out
a genome editing experiment.
So, for example, here, this is
a picture of Adrian Krainer,
a biologist who invented an
anti--sense oligo therapeutic
strategy against, I think it's
spinal muscular dystrophy.
So these patients will
succumb to the disease
at a very young age.
But he was able to rescue
that disease phenotype.
And these patients were
able to walk rather than
being paralyzed in wheelchairs.
And so this researcher's really,
really happy about the outcome.
So in those kinds of
situations, it's understandable.
Even if the editing outcome
is not a perfect fix,
it's a workaround.
But it was able to-- it
was able to alleviate
a really detrimental situation.
So this stands in
contrast to the situations
where the editing
was mostly geared
towards some kind
of enhancement,
so as a cosmetic editing
where people were trying
to gain better attributes,
physical attributes,
or something from the
editing experiment.
And so these-- it's hard
to gain ethical approval
in today's atmosphere.
So essentially, if it's
a treatment strategy
against a serious
disease, and it only
involves somatic editing, the
current ethical background
is that, yes, please proceed
with the therapeutic approach,
but with caution.
But it is an enhancement
type of editing,
the common understanding
is that you should not
have proceeded it in any way.
And germline editing
should not been proceeded
because of the safety concerns.
So that's the common
understanding.
But we'll discuss some
violations and situations.
So in the next few slides, I'm
going to basically give you
an overview of where
the industry is,
kind of a cutaway
of the forefront
of the CRISPR industry.
And it sounds like I'm
doing advertisement
for this industry, but
I'm learning it myself.
And so I'm stealing slides
from the company website.
So I think these are
the appropriate use
of the CRISPR technology.
So it appears that the
early pioneers, each one
basically set up
their own companies.
And so George Church
is this guy at Harvard
who has maybe hundreds
of different companies.
And so one of them
is called eGenesis.
And this involves
doing genome editing
on pigs with the ultimate
goal of maybe using
these pigs as organ
donors for human patients.
And so it's well understood
that organs are hard to come by.
And a lot of patients
die in waiting
for the organ transplantation.
And it's well known that
pigs has a physiology that's
very similar to human.
Also they're a great target.
But they-- you can't
just take the pig organ
and put it into human.
And they will be
rejected very soon.
So there are a lot
of surface markers
that has to be removed
in order to have the pig
organ recognized as a self organ
rather than a foreign object.
So that involved using
CRISPR tools to knock out
these kind of markers.
And pigs have some
viruses that are
potentially harmful for
humans, like retroviruses.
And so they're basically
diligently removing
these viruses one by
one from the pig genome.
And so initially it was
claimed as that they
have a very ambitious goal.
They were trying to reach the
human trials within two years.
That was 2016 or
something like that.
I haven't heard anything
from them on human trials.
So I think they're still
doing animal testing
maybe first in big animals,
primates, or something
before it's considered safe
enough to try on humans.
So another company is set
by Emmanuel Charpentier
who co-published that seminal
paper with Jennifer Doudna.
And so her company is
called CRISPR Therapeutics.
And they seem to be the
first one who came out-- roll
out the human clinical trials.
And so first CRISPR
clinical trial, and this
involves ex vivo somatic genome
editing using CRISPR/Cas9.
And it's a very popular target.
And so many companies are
targeting this one, sickle cell
anemia.
So if you studied
biochemistry, it's
a textbook example of
a molecular disease.
So the phenotype is
that the patient's cells
have this characteristic
sickle cell shape--
sickle shape.
And this is because of a
single nucleotide mutation
in the hemoglobin gene
that changed the identity
of a single nuclear acid--
a single amino
acid, which causes
essentially an aggregation
of hemoglobin in the patient.
And this is the first
molecular disease
being deciphered by researchers,
so by Linus Pauling and others.
So a perfect fix
would be to convert
this mutated nuclear acid back
to the wild-type sequence.
But because I
mentioned that Cas9
is good at destroying things
rather than fix things
precisely, this is hard to
achieve in a precise fashion.
So most of the companies
try to find a workaround.
So this company, the workaround
was based on this observation.
So, each human being has
two different versions
of hemoglobin genes.
So we have the adult version
and the fetal version.
So at birth, there
is a transcription
switching going on.
The adult version
is switched on,
and the fetal version of
the gene is switched off.
So in some
individuals, this gene
persisted and kept expressing
in adult individuals.
And there's only--
there's essentially not
much detrimental phenotype
in those individuals.
So that was the
therapeutic strategy
used by CRISPR Therapeutics.
And so their reasoning
is that rather
than targeting the
diseased allele
and trying to achieve a perfect
fix or a gain of function
rescue, we could target
this repressed allele
and try to essentially
disrupt the genetic--
disrupt the regulatory circuits
that shut off the allele
and allow the gene to express
in a constitutive fashion.
So what they did basically
was based on this paper,
they disrupted the transcription
factor binding site
and allowed this gene,
the fetal version,
to express constitutively.
And that was-- and so they
were able to rescue the disease
phenotype at the cell level.
And so they're now
testing it in humans.
And this is not a
naturally-occurring mutation
in humans cells.
And you don't want it to pass--
to be passed to the
next generation.
So it makes sense that they're
doing somatic genome editing.
And to be more prudent,
they're doing it ex vivo.
So they're extracting the
bone marrow from the patient,
and they're going to do the
genetic experiment in test
tubes, amplify the bone marrow,
and then for the patient,
they're going to basically
eradicate all the bone
marrow cells in the
patient and then
infuse the edited cells
back to the individual,
and basically undergoing a
bone marrow transplantation
but using the individual's own
cells that have been modified.
So everything seems
to make sense.
And so hopefully, they
were able to achieve
a cure for these patients.
So here is another clinical
trial that has just
been rolled out two days ago.
And this is another
pioneer from John's Lab.
He has this Editas company that
attracted a lot of attention.
And they have a long list
of therapeutic targets.
Interestingly, the
first one was a very--
it's a rare genetic disease
that has a pretty small patient
base.
But I guess they
targeted this disease
and put out the clinical trials
because it's considered a safer
therapeutic strategy.
So they're basically doing
localized in vivo human cell
editing.
And they're doing
it in human eyes
and targeting this
congenital eye disease.
So what I learned
from their website
was that basically
this is a mutation
in a structural protein
inside the eyes.
And this protein will cause the
collapse of the photoreceptor
cone.
So essentially, if not rescued--
if not fixed, this mutation
will cause blindness
in the patient within a
few years after birth.
And their strategy
was to deliver
an adenovirus containing Cas9--
encoding Cas9 and
guide RNAs locally
into the patient's eyes.
And the hope was that
this editing event
will rescue the function
of the deceased gene.
And we're going to get
the normal photoreceptor
cone and the normal
function of the eye.
And we're going to get
improvement from the patients.
So if we really look
at the-- while we
have a basis for
this therapy, it's
again a workaround from
achieving a perfect fix.
So here is the mutation.
And this is a mutation existing
in the introns of a gene.
So somehow, this mutation
generated a splice site
and basically causes apparent
splicing event that introduced
a exon in the patient.
And this exon contains
a premature stop codon
that essentially caused the gene
to stop here rather than splice
and translate into the
full-length proteins.
And that causes the disease.
And so the rescue was to not
directly fixing the mutation,
but use Cas9 programmed
with a pair of guides
targeting sequences
around the mutation site,
and try to generate
a double cut here
and trigger either a deletion
event or an inversion event.
In either cases this is going to
basically prevent this mutation
as being recognized as an exon.
This is going to prevent the
inclusion of a bearing exon
here.
And that will cause the normal
splicing and a normal function
of a gene.
So again, this is not a
mutation naturally occurring
in the human population.
And that's why they choose
to do somatic genome editing.
But this is the only
allowed editing anyway.
But it makes sense.
And it's a localized injection,
so we're not exposing patients
to unwanted editing events
in other places in the body.
So everything seems to
be rational in design
and makes a lot of sense.
So there are a lot of
considerations into the ethics.
But there are also cases
where it was not well thought
through, and there was the
poor way of executing science.
And essentially because
the tool is so easy to use
and is so potent, when
it's in the wrong hands,
it really causes
unwanted outcomes.
So we've heard in the
news that people start
to mail order CRISPR kits.
And we've heard
of people claiming
that they're going to do
garage genome editing and so
essentially, make
them more muscular.
And that's what--
I saw a video online.
They're like, I'm going
to edit my muscles
and I'm going to be-- and I
don't have to work out as much.
But nothing was more perturbing
when at the end of last year
when news came.
So there was a real editing
experiment in embryo
that causes the--
done by He Jiankui,
a scientist in China.
Essentially, this led to the
actual birth of a twin CRISPR
baby, so something that
people have feared,
and the scientific
community has been
self-policing to avoid doing.
And someone just
pushed the envelope
and did it all in once.
So that was very perturbing.
It caused an outcry in
the scientific community,
and including the
scientists in China
because they don't
know this guy.
This guy was not
a CRISPR expert.
And so he just came out of
blue and did the experiment
and thought that he's
doing something heroic.
So let's review the guidelines
for doing those genome editing
experiments in germ lines.
So there are two
versions of regulation.
And one is a 2015 statement from
the first human genome editing
consortium essentially.
And it's hard for me to read.
Basically, recognize the
limitations of the technology
and said that they
should never have been--
it would not-- it would be
irresponsible to proceed
with any clinical use
of germline editing.
But it relies on the
scientists to self-police
and it left on back doors.
So it says that we're
going to revisit the issue
and see whether it is more
acceptable in the audience--
in the general public, and
whether the technology becomes
safer.
And there is another
statement in 2017
by the US National
Academy of Scientists.
And so basically this
is a panel discussion
involving experts in the
US and representatives
from all over the world, like
major countries in China, UK,
Russia, and others.
So they came out with a
guideline for genome editing
experiments.
And so essentially-- whoops--
if you read the
guidelines and try
to follow these to design the
germline editing experiment,
none of the experiments would
have passed those guidelines.
It's very strict.
And again, it
relies on scientists
to follow the guidelines.
So the editing
experiments done by He
Jiankui was on this CCR5 gene.
So this is a gene that's
basically used by--
there's a surface receptor gene.
And the HIV targets this gene
as one of its co-receptors
to gain cell entry.
So interestingly, there was
a naturally-occurring allele.
And especially the
allele frequency
is higher in
European population.
And so this mutation,
CCR5 delta 32
is an out-of-frame mutation
that will cause a premature stop
codon and essentially
a truncated protein
being expressed.
And this protein can
no longer interact
with HIV surface markers.
And HIV, it can now gain
entry into the cells
if the individual is
homozygous for CCR4 delta 32.
So if you have one wild-type
allele and one mutant,
they can still use
the wild-type allele.
A virus can still
use that to enter.
Double mutant seems to
be immune against HIV.
And there was-- this was
discovered in a serendipitous
situation when an HIV patient
received a bone marrow
transplant from a donor that
was essentially a homozygous--
carries a homozygous
mutation for CCR4 delta 32.
And it was found that not
only did it cure his leukemia,
but also cured the HIV.
So one can imagine that in
certain disease situations,
it is a good target maybe for
genome editing experiments.
But nowadays there
are so many ways
to prevent the
transmission of HIV.
So if one uses the
genome editing of CCR5
as a preventive treatment, then
the ethical background for that
is very shaky because there
are so many alternatives.
And it's considered
not necessary.
So this news leaked out two days
before the second human genome
editing meeting.
And because of the news
attention, it was--
his talk was broadcasted online.
And I was among the 1 million
viewers around the globe
basically watching and
hearing what he said.
So these are the reflections
I remember at the time.
And it gave me the impression
that he really did it.
And there was a
situation where--
the scenario was that the
dad was the HIV positive,
and the mom was negative.
And so in such situations
when actually the transmission
of HIV can be
prevented in many ways,
including washing the
sperm before the inception
and fertilization, that would
be usually sufficient to prevent
the viral infection.
And so then it really
begs the question,
where was the
unmet medical need?
So there is just no need to do
this genome editing experiment.
And then the next
question is, how did it
achieve the clinical--
the ethics evaluation?
How did it pass that?
How did it-- how was he able
to achieve the approval?
And I mentioned
that in most cases,
the editing outcome is
messy and unpredictable.
And in this case,
it really showcased
that this is the case.
One should not have
used it very lightly.
And so it was a twin
girl being born.
And one of the individual
carries mutations
in both alleles of the gene.
It kind of mimics the CCR5
delta 32 mutation, but not
the exact mutation.
So it's not the
exact delta 32 fix.
But it mimics the premature
truncation situation.
The other individual has
one wild-type allele.
And the other allele carries
just a short deletion.
And it didn't fully
knock out this area.
And so this particular
unnatural variant
probably would not have
conferred HIV resistance.
And with the wild-type
allele, it definitely
will not provide resistance.
So at least for this individual,
it's a failed experiment.
And for this one, there's
unpredictable fitness
consequences.
So in both cases,
I would say it's
not-- it's a very shaky
experimental outcome.
And it really begs
the question--
the further questions,
so these two individuals
never signed any consent forms.
And they were brought into the
world without any consultation.
So how do we protect
their own interests?
And how do we prevent
similar reckless attempts?
So there are a lot of
discussions going on
in the community, including
talks about moratoriums,
I guess.
But they're opinions
from both sides.
And so this is being
heatedly debated right now
in the scientific community.
And so that study still
has rippling effects
after half a year.
And so this is a new study
coming from Nation Medicine
just last month.
And so essentially
talking about the fitness
consequences of having the
CCR5 delta 32 indentation.
And so as a
statistical analysis,
basically the observation
was that individuals
with homozygous mutation
tends to be under-represented
in senior population.
So the interpretation
is that maybe there's
a fitness consequence.
Their life expectancy is not as
long as a wild-type individual.
So there might be a fitness
cost for being HIV-resistant.
And so there was an early
study published in Cell
claiming that there might
be some beneficial effect
from having this mutation.
So the observation
was that when there's
brain damage, for example,
a stroke, in a person,
a human being with the CCR5
mutation is more resilient
and seems to recover
with a better outcome.
So it seems like there is some
gain of function phenotype.
Then it becomes-- so you can
interpret these scenarios
in different--
with different sentiments.
So, for example, if we
use genome editing really
liberally, and so
we inadvertently
generated some gain
of function phenotype,
some genome editing outcome
was a super human kind
of a phenotype, then the
average person becomes--
has to undergo this debate.
Are we under pressure
to also adapt?
So there are some ethical
concerns there as well.
So it just underlines
the importance
that we really
need to understand
the outcome of the
editing experiment
before we do anything seriously
with clinical research.
So I don't know a how--
what was the-- how many?
MAN: You're OK.
Go ahead.
AILONG KE: OK, good.
So I want to spend
maybe 10 minutes talking
about my own research.
[CROWD CHUCKLES]
So the question is, there
have been 10 years of CRISPR.
Is it-- it has been at the
forefront of a revolution
in biology.
And so the question
is, is the fever over?
Certainly from my perspective,
working on it day by day,
I feel that we're
still in the wave.
So every time we think
that we're over the peak,
there are new and very
interesting biology coming out.
So, I mentioned
that there are a lot
of diversity in CRISPR systems.
And in the early days, there
are three different types.
And so by now, there's
six different types.
And so we can really
grouped them into--
the CRISPR system into
two major classes.
And the star molecules in
the genome editing fields,
the Cas9s, the
Cas12s, or Cas13s,
they really belong to one
class of CRISPR systems.
And these are the single
component CRISPR systems.
And so they're easy--
they're very simple,
one-effector protein,
in many cases, one guide RNA.
So it's easy to adapt them
for human genome editing.
So there are yet another class
of CRISPR systems that involve
multi-component effectors, so
more sophisticated CRISPRs that
are hard to adapt but carry--
nonetheless carry very
important-- very interesting
attributes.
So the major differences are,
I would argue, the nucleuses.
So they basically use the same
principle to look for targets.
But the nucleus have
different functions.
So in Cas9-- in single-component
systems, usually
the nucleus stop at making
a double-strand break.
But in systems like type
1, for example, the nucleus
is almost like a terminator.
It destroys everything
after targeting.
And so by now, we understood--
so the first step of
CRISPR interference
involves cutting a piece
of DNA and inserting
into the CRISPR array.
10 years ago, it was somehow.
And nowadays we have
a good understanding
of the molecular mechanism.
My lab was one of the labs that
contributed to key insights
into its molecular mechanism.
And in type I system,
the effector complex
is assembled from five
different proteins.
And this really is a beast,
like 400 kb, large complex,
multi-component
systems, recognized PAM,
opens the R-loop,
similar principles.
But what's different
is what happens
after the R-loop formation.
So in type I system, the
actual nucleus is a--
it's a very
sophisticated enzyme that
has two enzymatic activities.
Not only does it
have the nucleus,
but they also have
a helicase activity.
So basically it's a nucleus
fused with a locomotor
so it can move.
And so this nucleus
is recruited in trans
to the R-loop-forming cascade.
And once recruited
and activated,
it's going to cleave the
single-stranded DNA not
base pair with the
guide RNA, and then
fire its engine at the
expense of APB hydrolysis,
and erase the DNA for a
long stretch of distance.
And maybe that's
the reason why this
is the most popular
CRISPR systems in nature.
Because once targeted,
it really leaves
no chance for the parasitic
elements to repair.
It erases the genetic
information completely.
So that's basically-- it's
going to generate a much
bigger impact upon targeting.
And so this has been
the focus of my lab.
And so as a biochemist
and structural biologists,
we really want to
understand the mechanism
and provide the highest
resolution information
about the mechanism.
And essentially, we're
trying to generate
snapshots of these complexes
in different stages of action.
And part of it is serendipity,
but a part of it is hard work.
We're able to achieve a lot
in this particular system.
And so essentially, we
solved the structure
of the cascade, the
effector complex,
and it is binding to
double-stranded DNA and opening
an R-loop mimic.
And we solved the
nucleus, basically
a helicase, a
nucleus-fusing enzyme,
and we know how it
recruits the DNA
and degrades it persistently.
And by applying the cutting
edge structural biology tools,
something called Cryo-EM, we're
able to capture the cascade
in motion, capturing
different snapshots
of this complex binding to
DNA, opening an intermediate,
and opening the entire
R-loop, and then
recruiting the nucleus
forming the ternary complex,
and then nick the DNA,
and start to carry out
its job to destroy it.
So really hard biochemisty,
and the resolution determines
what kind of a mechanism--
how reliable the
mechanism can be.
And with the
resolution, we're pretty
confident that
we're really diving
into the heart of
the mechanisms.
And four different snapshots.
And so the mechanism is this.
So essentially the
goal is to scan
for a match that's
complementary to the guide RNA
and eventually open
the R-loop and recruit
a nucleus to cleave it.
And so the process involves--
essentially the start
of the R-loop formation
involves the cascade
holding the DNA
and really bend it to a
very uncomfortable situation
and trigger the DNA to undergo
a DNA bending and melting
transition.
So you force it to
transiently breathe.
And so that moment of breathing
gives the cascade effector
a chance to check
whether the target strand
DNA is complementary to
the guide RNA or not.
So if it's not,
then it's rejected.
If yes, then it's stabilized
into an intermediate.
And then the cascade
attempts to further unzip
the DNA, very much like
when we unzip our zipper.
So it's a directional
DNA unwinding process.
When you unwind
the DNA, you need
to encompass it
for the energy loss
by forming base pairs
with the guide RNA.
So then the entire process
become very smooth.
And when the entire
sequence checks out,
it enters into a
point of no return.
And so then at that point, a
lot of things happen at once.
And the cascade is stabilized
into an R-loop forming
confirmation.
So these seriously
events involves
a confirmational change in the
cascade that essentially locks
down both strands of DNA,
a single-stranded form,
and then reorganize the surface
in one area of the cascade,
and also traps a flexible bulge
near that reorganized surface.
So this sends a signal
for the nucleus to bind.
And so this nucleus is
the sequence non-specific.
And so it only cares about
whether the effector complex
has found the target or not.
And so once it
finds the target, it
generates that
signal, then a binds.
So once bound to the cascade--
so that flexible bulge
allows the nucleus
to swallow the substrate and
makes a nick in the substrate.
And thus nick then spontaneously
repositions the substrate
into a position that essentially
allows the helicase to thread
the single-stranded
DNA through the nucl--
through the helicase
into the nucleus.
So then this Cas3 enzyme is in a
position to fire as the engine,
burn ATP, and start
to [? attracting ?] it
towards itself, and
eventually lead the cascade
and move by myself
for a long distance.
Along the way, it's going
to chop the DNA to pieces.
So that is the
mechanism from years
of biochemistry and
structural biology.
And we're quite
confident about it.
And so then the next goal--
at some point, we were like,
we know enough about it,
and so maybe we can
test it in human cells
and see now what we can do--
what kind of a genome editing
we can do in human cells,
and whether we can come up
with creative applications
from that.
So I started to go to a
different genome editing
meetings and hear about
all the progresses.
In one of the meetings,
I met this brand
new assistant
professor Yan Zhang,
who just finished training
with Erik Sontheimer
and started her own lab at
the University of Michigan.
And she saw through
me, and she said,
you must be thinking about
doing genome editing in type I
system.
And so I have this
perfect system.
We should collaborate.
And so it was a very, very
productive collaboration.
And so what we
provided from our lab
are good mechanistic
understandings and a blueprint
for how we should deliver the
complexes in the active form,
how to target it to the nucleus
where the DNA targets are,
and what kind of a
genome editing outcomes
we might be anticipating, and
how we should detect them.
And from Yan Zhang's side, they
have a great editing platform
that we could use,
and all the expertise
in doing genome
editing experiments
with other Cas9 proteins.
And so in her platform,
the equivalent
of a chromosomal
loci, in one side,
she tacked with GFP,
green fluorescent protein.
On the other side,
she has essentially
a red fluorescent protein
on the other side.
So then why do you
target the GFP,
and you inactivate
the GFP signal,
the red fluorescent protein is
serving as a internal control
for the experiment.
And so the editing outcome--
so her system is really,
really sensitive.
And it's the normal cells,
not the cancer cells.
And so we're really dealing
with normal somatic cells
and looking at the outcomes.
So with good understanding, that
really translates to success
from day one.
And so the first editing
experiment just worked.
And we saw-- in the yellow
background normal cells,
we saw the appearance
of red cells.
And that really becomes--
so it only appears when we
deliver both the targeting
complex and the nucleus.
We deliver individual ones,
they're not effective.
And this is our cell
sorting experiment.
We're monitoring both the
green fluorescent signal
and red signal.
If it's an unedited
cell, the cells
should populate along
the diagonal lines.
If we inactivate
GFP, we're going
to see accumulation of red
cells at this quadrant.
And so that's
exactly what we saw.
And this is, again,
programmable.
If we target tdTomato, the red
fluorescent protein we saw,
green cells are accumulating.
And it's very obvious
that the activity
is limited by the target
searching complex, the cascade.
And so the more
cascade we deliver,
the more activities we saw.
And so these are old data now.
So we're seeing 13% editing,
meaning that every 100 cells we
saw, 13 cells being edited.
And now in some genome locus,
we saw editing efficiencies up
to 60% to 90%.
And so if there seems
to be some dependency
on the chromosomal environment.
And we still don't
fully understand
exactly how this plays out.
And the outcome was
quite different from what
one saw for Cas9.
So this is a genome
editor that will really
impact a long stretch of DNA.
And so this is
the targeted site.
And this will be the direction
of the Cas3 helicase movement
direction.
And we're seeing directions--
we're seeing divisions along
the Cas3 movement direction.
And the start up the deletion
is a little bit random.
But it's always upstream
of the targeted site.
And it ends in the
random position.
But in almost every case, we're
deleting a very long stretch,
like kilobases of DNA.
And so when we apply deep
sequencing detection method,
we're seeing that on average
most of the deletions
involve maybe three to five kb.
And keep in mind we're
doing RNA protein complex
injections rather than
delivering RNase and allowing
to amplify inside.
So if we were to deliver a
lot more RNPs or repeated
deliveries, we're going
to see very long deletions
along this line.
We're already seeing the
deletions up to 30 kb,
sometimes 100 kb.
So this is impacting genome
in a very profound way, so
a true long range DNA eraser.
And so it may not be a perfect
scenario for precision medicine
at this point, but for a
research point of view,
98% of our genome are
non-coding sequences.
And there are many
important genetic elements
in these areas.
And we just don't
have an efficient tool
to look for those six elements
and understand their function.
And I think that
those CRISPR/Cas3 tool
is an efficient screening
tool to decipher
the non-coding genomes in
a high-throughput fashion.
And the fact that in a
single-targeting event,
the targeted site is
intact in most cases
allows one to essentially
repeatedly administer
CRISPR/Cas3s into the cells.
And we're going to get a
gradual increase of editings
and achieving
different outcomes.
And we are thinking about
therapeutic applications.
And some might be more
challenging than others.
But the most obvious
one we're thinking about
involves targeting essentially
ectopic viruses in our cells.
For example, a very famous
example is the herpes virus.
So these cells undergo two
phases of the infection.
And there's the acute phase
that causes sores, cold sores,
something like that.
And so the herpes is famous for
then entering into a dormancy
and basically hide
inside our nucleus almost
like a plasmid, a kind
of a circular DNA,
and just hide there,
and wait for the moment
when the host, the immune
system becomes weak,
and they really come out again
and cause a severe infection
again.
And there were some reports
claiming that herpes
in some cases cause lymphoma.
And in some other
cases, there is a link
with the Alzheimer's disease.
So all these things are ongoing.
A variant of the
herpes virus, something
called the Epstein-Barr virus,
undergoes the same latent
infection cycle.
And these herpes target
the immune cells.
And so there's a clear
link that the EBV
is causing lymphoma
in many patients
and causing nasal cancer
in other individuals.
And this nasal
cancer case actually
is pandemic in Southeast Asia.
It seems to be correlated with
a specific strain of the EBV
virus.
So in both cases--
so if you were to
target Cas9, you're
waiting for the repair enzyme
to make a mistake in order
to maybe inactivate the viruses.
And so in our case, if we
were to deliver our tool
into the cells, once
targeted, basically there's
no chance for
evasions of the virus.
And so these viruses will
be actively destroyed.
And so in theory, on
paper, we have a way
to erase those viruses
very efficiently.
And so there is
yet another case.
Hepatitis B virus,
so that exists mostly
as ectopic circular DNA.
And there's a clear link
to cause liver cirrhosis
and carcinoma.
And this is pandemic in
many third world countries.
And just a little bit
more dangerous to use it
against the HPV
because sometimes they
integrate into the genome.
But nonetheless,
these are viruses
that cause severe human
disease and could be
targeted by our genomic tools.
So this is a preview of
what's going on in our lab.
We're still trying
very hard to achieve
the potential of our tools.
I'll just stop here and
thank the people involved
in the work.
And so all the structural
work was done by this--
a single individual,
Yibei Xiao, in our lab.
And so previously,
Bob Hayes also
solved some important crystal
structures of the cascade.
And together, these two really
provided a very, very solid set
of mechanistic understanding
of the system that
allows the eventual
applications to take place.
And Adam Dolan is this smart
kid, undergraduate student,
who worked with Yan Zhang's
lab and demonstrated
usage of CRISPR/Cas3.
And I didn't have time to talk
about Sherwin and Jagat's work
on the acquisition part
of the CRISPR biology.
I was fortunate enough to
collaborate with many peoples--
many people in the past,
and so great collaborators.
I just want to give a special
shout out to the junior faculty
that I collaborate with
on the CRISPR biology.
And this includes the
EM specialist Maofu
Liao at Harvard and
single molecule biologist,
Ilya Finkelstein at UT
Austin, and then Yan Zhang
for genome editing at the
University of Michigan.
And funding was from NIH.
I guess I'll just stop
here and take questions.
[APPLAUSE]
Yes.
AUDIENCE: You mentioned
nasal pharyngeal cancer,
and Guangzhou in particular,
Southeastern Asia and China.
AILONG KE: That's right.
AUDIENCE: Well, one of the
things that's been spoken about
is the preponderance of
people using their cell
phones over there, which up
regulates the Epstein Barr
virus--
AILONG KE: Huh.
AUDIENCE: --at 50 Hz.
And if-- I have a home in
Dubai and spend a lot of time
in Hong Kong and
Dubai and that area.
Literally young Chinese
people in that area
live with their phones
connected to their bodies.
AILONG KE: That's an
interesting observation.
Although, I don't know.
So this has been pandemic
before the cell phone.
So I don't know.
There might be an enhancement
from those exposures.
But certainly beyond that, there
is some connections already.
There was pandemic in
the '70s, '80s, and so.
AUDIENCE: And in
your bio, you have
some training in mitochondrial
DNA and epigenetics.
You didn't think about
that at all tonight
and the relationship
between the 1,300 [? dB, ?]
and the mitochondrial DNA,
and the 24 RNA transcriptional
genes in the
mitochondrial DNA, which
we inherit all from our mother.
And I just want to
know how you feel
they are affected by this light
versus the natural sunlight
which we evolved in.
AILONG KE: Hm.
Well, to be honest,
I'm not an expert
discussing the electromagnetic
field, the effect
of this field on DNA.
Yeah, I think I should refrain
from saying too much of that.
AUDIENCE: They're not
affecting the DNA.
They're actually affecting
the software, which then
affects the DNA in genomes.
AILONG KE: Well,
let me read more
into that before I make a--
AUDIENCE: This is very narrow.
And managing your light
is between 440 and 475.
Our evolvement basically came in
natural full-spectrum sunlight,
280 to 700.
We're living in a much
different world than we did,
even our great-grandparents.
AILONG KE: I agree
with that, yeah.
So it's going to
affect our rhythm.
It's going to affect our vision.
You're talking about
affecting our transcriptomes.
AUDIENCE: Exactly.
AILONG KE: Yeah.
So that, I need to read more.
So that's something I didn't--
AUDIENCE: And it affects
germline and the preponderance
of autism that's happening
today in the world, too.
It can be passed down not
even multi--generational,
but maybe skip a generation
from grandma to grandchild.
AILONG KE: Right.
So yeah, wow,
that's fascinating.
I thought that the energy
was too low to cause that.
So let's read something
before I draw conclusions.
AUDIENCE: It's not an ionizing
energy, so it's not thermal.
AILONG KE: OK, yeah, please.
AUDIENCE: So, given all of
the pros and cons of CRISPR
that you described
here, are there
other kinds of techniques
that are being explored
that might have complimentary
or different [? reactives? ?]
AILONG KE: Right.
So pros and cons, which aspect?
Are you talking about
the accuracy part?
AUDIENCE: Yeah.
AILONG KE: OK, yeah.
So, in terms of
accuracy, I think so far
it beats all the other
alternative tools
on the market.
So even though it's not perfect,
it's better than other tools.
One thing I didn't
mention, our Cas--
so the Cas9 targets something
like a window of 20 base pairs.
The type I system targets
a much longer region,
about 30 base pairs.
So potentially,
it's more accurate.
So there are different
alternative tools that
are potentially more accurate.
And there are
alternative strategies.
For example, without causing
double-strand breaks,
you can fuse it with base
editors and chase DNA
sequences that way.
And so there was this
incredible creativity
among researchers trying
to make it a better tool.
So I think it's just
a matter of time
that we're going to
achieve that, yeah.
But so far no alternative
that's better.
Yeah.
AUDIENCE: That was
an excellent review.
Thank you very much for
the well [INAUDIBLE]..
Just a comment and a question.
You're absolutely right, NPC
has been around for many years
well before cell phones.
So the head and neck
surgeons, and ENT surgeons,
it's a real problem that is
well recognized to the EBV.
So if your system
can target that,
it will be a huge contribution
to head and neck cancers
that ENT certainly encounter.
But my question is this to
you, the ethical dilemma
that you described reminds one
of the 1970s when I read about
with recombinant
DNA technology--
AILONG KE: That's right.
AUDIENCE: --and the moratorium
that occurred in many places.
And then, because we need people
into [INAUDIBLE] and people
to legislate even knowing
what the outcomes would be,
do you think the solution's
going to be waiting
till it gets more precise?
Or do you think something
else has to happen
because there's so homologous to
what happened in the '70s with
recombinant DNA--
AILONG KE: That's exactly--
so when the recombinant
DNA technology just became,
and there was like
all those precautions,
and we're going to--
we should burn all the
DNA before we release them
into the environment,
that kind of thing.
So yeah, it's hard to say.
I think from the researchers,
I think the common agreement
is that right now is not so--
it's not safe not to
do germline editing.
That's the common agreement.
But people are trying.
I think in the end, it might
be that the therapeutic--
we'll accumulate data from
therapeutic approaches.
And it's going to reach
to a point that we,
with the follow-ups, we
build up our confidence
and to a point that
we're comfortable enough
to try and germline cells.
Because you can hear
arguments from both ways.
So somatic editing,
it's almost impossible
to achieve 100% editing.
So whereas if you were
to do it in germ lines,
if it's a perfect
editing, you only
need to worry about
two targets, and then
biology will take its
course, and the individual
will have perfect
genes in every cell.
So one could argue
both ways, that they
could be the perfect
solution, but right now it's
not safe enough.
Yeah.
AUDIENCE: Thank you for
giving a good lecture.
I was wondering, even
in somatic cells,
is there a possibility of
epigenetic inheritance?
Even though you're not,
obviously, a [INAUDIBLE] genes,
is there a way to
affect the environment?
Does that make sense.
AILONG KE: Yeah.
That's hard to
answer, I would say.
In most cases, the
epigenetics are reset in the--
when the-- in the embryonic--
in the embryos.
I would say probably to--
I don't see worries on that
too much at this point.
I think most people
are either doing it
ex vivo or localized in vivo.
There are some efforts
on the whole body,
trying to achieve that.
But I didn't hear any
clinical trials on that yet.
So even that I think people
are perceiving with caution
at this point.
Yeah.
AUDIENCE: I'm sure this is
not you're slice of area,
but in terms of the safety
of these kinds of approaches,
do you deal with
myeloablative conditioning?
There's actually a risk of
mortality just from that.
So it would seem that
[INAUDIBLE] to risk,
germline therapy might
seem more attractive.
Your thoughts?
AILONG KE: Yeah, I agree.
That's a painful
procedure to go through.
And that has the
potential possibility
to cause cancer and increase
the risk of cancer, I would say.
Yeah, so I've read
it in both ways.
So if the germline editing
can be done in the precise way
without the possibility
of off-target effects,
then it could be the
perfect solution.
But the technology
is not there yet.
And the public is
not with us yet.
AUDIENCE: How do you mail
order a CRISPR package?
And how much does it cost?
Can I get one at Amazon?
And how do I use it?
AILONG KE: Yeah.
So if you spend $60, you
can get it from Addgene.
But you have to purify
yourself, I guess.
Well, you can get
it from a company,
that really comes in mail.
But yeah, it was a joke.
So I'm sure these
experiments will fail.
That's why I'm not
so worried about it.
It's the one that--
the educated person who ignored
all the ethical considerations,
that was the most
worrisome part of this.
AUDIENCE: Is it
legal to sell them?
AILONG KE: I didn't
check that yet.
So I should check it before
I answer that question.
AUDIENCE: OK.
AILONG KE: So any other people
sell various stuff on eBay,
so I don't know.
AUDIENCE: Yeah,
I'd be interested.
AILONG KE: Yeah.
Oh, seriously?
AUDIENCE: Yeah.
AILONG KE: OK.
AUDIENCE: Who else
has any questions?
MAN: [INAUDIBLE].
AUDIENCE: No?
OK.
MAN: Cool.
AILONG KE: OK, thank you.
[APPLAUSE]
ANNOUNCER: This has
been a production
of Cornell University on
the web at cornell.edu.
