1. What are you researching specifically with CRISPR-Cas9?

a. Since CRISPR-Cas9 is efficient, it can be used for a multitude of

scientific/biological reasons, and it can be developed into a tool for editing the
genomes of organisms. Right now, I am working on figuring out a way to deliver
Cas9 to a specific organ. Currently, if you want to edit something youll have to
edit the entire genome of the organism. Secondly, we are working on delivering
Cas9 to a compartment of a cell.
2. What are your biggest challenges when working with CRISPR?
a. One of the biggest challenges is finding a way to trick the cell into
letting Cas9 enter it. Cells have a natural defense mechanism that does not allow
foreign substances to enter it, and we are trying to find a way to bypass that
mechanism so that Cas9 can permeate the membrane and edit the cells DNA.
3. Have you found a way around these challenges? If yes, how?
a. No, not quite yet. But we are researching ex vivo delivery of Cas9,
which is when you take the T-cells from a person, place them in a petri dish with a
Cas9 solution, let Cas9 edit them, and then inject them back into the person.
4. What exactly does CRISPR do to edit genomes?
a. So CRISPR-Cas9 is a system that was actually discovered in
bacteria as an immune system. Basically, Cas9 is an enzyme that cuts doublestranded DNA at the targeted gene. Then, the cell will repair the damaged
chromosome, which is really just wrapped-up DNA, and will destroy the cut gene
in the process.
5. What methods do you use for delivering Cas9?
a. Well, as I said before, the cells would be put in a petri dish that
contains a Cas9 enzyme solution. Then we would give those cells an electric
shock to let Cas9 in. Also, engineering tiny pieces of viral protein assists with
sneaking Cas9 into the cell.
6. When do you think CRISPR will be applicable to most of the medical field?
a. You know, will it ever be acceptable in the medical field, and thats
a question of safety, right? We havent really had any CRISPR therapies take
place and be assessed for safety, I mean there are some preliminary trials going on
right now, but I think it might be maybe 10 years before were very convinced
that its safe enough to be used in a widespread way. But, um, yeah, i think that
safety will probably be established. So, the two issues with this one is establishing
safety and doing lots of testing. So both of these things will take a lot of time and
I think ten years is enough to make sure that its safe and for us to have done a lot
of test to make sure its not causing any side effects that we dont want to take
place. One kind of promising thing is that there are some genetic diseases that are
so debilitating and lethal that for these people, their life is already threatened, so
even if there is a little chance of side effect from a CRISPR based therapy, I think
it will be beneficial for these patients to have this therapy because itll give them a
chance to survive. And, um, in the case of these people, it will be really helpful

because for them to gain any chance of improvement in their survival, I think
CRISPR will be a welcome therapy, and through these people well be able to see
if any side effects arise. I think its very likely that CRISPR will not have side
effects, its just a question of you know, how do we test that and how do we make
sure that people will be okay. But yeah, we can a lot of testing in animals, and I
think there will be very sick people who will be the first recipients of CRISPR
therapy. And I think thats where were going to learn a lot about any extra risks
that come with it.
7. How do you think CRISPR will be used in the future?
a. Yeah, so I think some of the first uses will be used in ex vivo
therapy, so in this case youd take some cells out of the patient and um, alter them
using crispr and put them back into the patient. These cells can be changed to do
amazing things, like target cancer cells specifically. So you can actually take cells
from the immune system and change those so that they can become hyperactive to
target cancer cells. So, thats one very clear therapy. Theres also a lot of promise
for muscular dystrophy, where theres a lot of genetic basis for that disease, and
theres already been some preliminary results showing that in a mouse that has it,
you can remove, so of like, cells from that mouse and return them to the mouse,
and the mouse will have improved muscle function. So, um, I think for severe
diseases that are caused by genetic defects, like muscular dystrophy, theres going
to be some exciting promise there. And, um, theres also going to be work to
genetically alter animals, like pigs for example, to make them non-immunogenic,
and I think the goal there is to create a pig, for example, that can have an organ
that can be transplanted into a human. And it [the human] wont have a negative
immune response. This will be a huge leap forward, and its something that
wouldnt be possible before this CRISPR-Cas9 technology, really. You need to
make a lot of genetic edits to the animal to make it suitable.
8. What are the risks of commercializing CRISPR for use in the medical and science
fields?
a. Um, I think the main risks are the safety issues that I already talked
about I dont think I can come up with any other risks off the top of my head
um yeah, I think I talked about the main risks thats about safety, but Im not
sure if we have any adequate regulation right now, honestly. So, perhaps if we
dont do a good enough job of regulating, there could be issues around that. I
think its a very easy technology to use, so I suppose people could be creating
genetically modified animals, you know, without any oversight, and there could
be some environmental consequences there. You know, I think youve heard of
these mosquitoes that have been engineered in the hopes of decreasing Zika
transmission. So these companies can make an animal and release it into the wild.
And this animal could go into the wild and interbreed and actually devastate the
population. So I think irresponsible use of this technology can actually have some

adverse effects and consequences, but i think reasonable regulation can decrease
these risks a lot. I think theres a reasonable level of regulation going on, there
shouldnt be too many risks once we establish safety in humans, for example. I
think things like agriculture, or editing plants or animals, which is something
where things could slip through the cracks. I think there are regulations for you
know, human related therapies, but for plants and animals I think thats one place
where things could slip through the cracks.
b. [Alyssa]: Like I think I heard that, um, theyre planning on wiping
out the mosquito population, like the ones that transfer Zika?
c. Right. Theres um, a push, or a program to release these sort of
mosquitos but I think that thing has been designed in a way that its short term, so
I think those mosquitos arent gonna leave a lasting impact on the environment.
But I think, if Im not mistaken, maybe its just changing those mosquitos so that
they cant have the Zika virus? [laughs] Yeah, you should probably read up on
that.
9. Will CRISPR be too expensive to use if it cannot be commercialized?
a. Um, I think that for certain uses it will be affordable. Its certainly,
um This is an interesting question um, I think that the time when its very
easy and cheap to use CRISPR is when something is a single cell. So, the good
news is that for editing plants and animals theres no ethical concerns here, and if
you want to alter a plant or animal you just do it at the single cell stage and youre
set. But for human editing, this is a huge ethical concern, so to do it at the single
cell stage, that means two things, right? Youre making a change to a person who
isnt agreeing to that change, and second youre creating a person whos going to
pass that change on to their children. So I think the big goal right now is to do
what I call ex vivo editing where you take cells out of a patient, edit them, and put
them back, and there are some other places where you can do edits to the eye
specifically, and there have been degenerative diseases that have been targeted
specifically with Cas9 therapy. And I think in these casees, yeah, its not
prohibitively expensive. But to do genetic therapy to a patients whole body, it
could get pretty expensive, admittedly. Yeah, I think it could be tricky, but a lot of
these ex vivo editing is pretty complicated to carry out because you have to keep
the cell sterile the whole time, and um, theres a lot of labor involved with that. So
in the beginning I think CRISPR is going to be used for a lot of really severe
diseases, and even though people like to talk about these kind of fun, cosmetic
improvements, I think those are a ways off just because its not super trivial to do
this stuff.
10. If off-target mutagenesis occurs unexpectedly, is there a way to reverse it or edit
the gene to make it normal again?
a. So, I think anytime you make an edit to the gene theres some
small risk that youre going to make an off-target edit. The problem with trying to

correct this is that its gonna happen one in ten thousand cases. So if you have one
out of your ten thousand cells that has this funny edit, um, even identifying that
edit will be very hard and even correcting that edit will cause the same problem
all over again. So even if you run the risk of fixing the gene, youre going to make
another accidental edit somewhere else. So, I think the question is whats the
frequency of these off-target effects and how often will this cause any problem for
someone. I think cells experience this kind of DNA damage all the time and
theyre constantly repairing themselves, so theres a chance that the off-target
effects caused by CRISPR-Cas9, um, are really just static that blends in with the
DNA damage thats constantly happening within cells. Theres a pretty good
chance that these off-target effects are just going to be business as usual inside the
cell. So I mean, to be frank a big risk is if you had an off target effect that
somehow led to a cell, you know, developing into a cancer cell and becoming a
tumor, then at some point it becomes a question of treating cancer. Its not a
question of fixing an off target effect. So, thats the kind of worse case scenario.
And, you know, thats sort of what happened in an old study, so, um Theres
this disease called skid, that has to do with immunodeficiency, and this was
attempted to be treated with gene therapy in the early 2000s, and they saw with
these patients that some of them ended up getting leukemia because of unwanted
genetic changes. So we have to make sure that the CRISPR-Cas9 therapy is not
going to cause these changes. And so far, you know, there have been a lot of
animals who have been edited with CRISPR-Cas9 and theyre fine. We just have
to make sure that this is not a likely issue, yet.