CRISPR stands for "Clustered Regularly Interspaced Short Palindromic Repeats," a system in bacteria that acts as an adaptive immune mechanism by identifying and cutting foreign DNA.

How does the CRISPR-Cas9 system work?

The CRISPR-Cas9 system uses a guide RNA to identify and bind a specific DNA sequence, where the Cas9 protein then makes a cut, effectively disabling the targeted DNA sequence.

What are some potential applications of CRISPR?

CRISPR can be used for gene editing to correct mutations, develop treatments for genetic diseases such as sickle cell anemia and cystic fibrosis, and even modify organisms like mosquitoes to control disease spread.

What are the ethical issues surrounding CRISPR?

Ethical issues include the potential for designer babies, accessibility, control over gene editing rights, and the implications of modifying human embryos.

What are the limitations of CRISPR technology?

Limitations include the challenges of delivering CRISPR into human cells, potential off-target effects, and the unpredictability of DNA repair post-editing.

What is the significance of the PAM sequence in CRISPR?

The PAM (Protospacer Adjacent Motif) sequence is crucial for identifying and cutting the correct DNA sequence, preventing the system from cutting its own DNA.

Can CRISPR be used in cancer treatment?

CRISPR holds potential for cancer treatment by potentially correcting mutations in tumor suppressor genes, though research is still ongoing.

What role does Cas9 play in the CRISPR system?

Cas9 is a protein that binds to the guide RNA, recognizing and cutting specific DNA sequences as dictated by the CRISPR process.

Can CRISPR be used to create vaccines?

While CRISPR's use in direct vaccine creation is uncertain, it can potentially be used to alter immune responses or identify viral components for diagnostics.

How does CRISPR relate to epigenetics?

CRISPR can potentially be used to demethylate DNA regions, affecting gene expression, although this application is still under exploration.

GRCC Science Talk: Gene Modifications and CRISPR by professor William Faber

00:58:34

https://www.youtube.com/watch?v=Ov9jpLzFNbc

Zusammenfassung

TLDRThe presentation introduces the CRISPR-Cas9 gene editing system, highlighting its use in identifying and cutting DNA sequences in a manner akin to an adaptive immune response in bacteria. Originally derived from bacterial defense mechanisms against viruses, CRISPR's ability to edit DNA has been adapted by scientists for broader applications including treating genetic disorders like sickle cell anemia and cystic fibrosis. The speaker details how CRISPR recognizes DNA sequences using a guide RNA combined with the Cas9 protein to make precise cuts in the genome. However, challenges such as effective delivery into human cells and ethical considerations around genomic editing remain ongoing discussions within the scientific community. The talk also touches on potential uses in diagnostics, agriculture, and disease control, with a focus on balancing technological promises with moral constraints. Key points include the fundamental structure of DNA, RNA, and proteins, and how CRISPR can be an advanced tool for gene disruption and modification.

Mitbringsel

🧬 CRISPR-Cas9 is a gene editing tool derived from bacterial systems.
🔬 It uses guide RNA and Cas9 protein to cut specific DNA sequences.
🧪 Offers potential treatments for genetic disorders like sickle cell anemia.
🤔 Faces ethical debates over the implications of genetic modification.
💡 Originally a bacterial defense mechanism against viruses.
💊 Promising tool for genetic diagnostics and potential therapies.
📈 Has significant implications for agriculture and healthcare fields.
🔍 Ongoing development needed for safe human applications.
🛠 Combines biology's understanding of DNA/RNA with biotechnological advances.
🚫 Potentially controversial applications include designer babies.

Zeitleiste

00:00:00 - 00:05:00
The speaker begins by expressing gratitude to Tim for organizing the talk and mentions their background in DNA arrays and recognition. They introduce their interest in CRISPR, despite not being a CRISPR scientist, and highlight its role in identifying unique DNA sequences, touching on their own experience in diagnostic biomolecule analysis.
00:05:00 - 00:10:00
The presentation will cover: adaptive immune systems, applications of CRISPR-Cas9, and its limitations. The speaker outlines basic concepts about DNA, RNA, and proteins, noting that proteins perform essential functions in the body. They also briefly describe the structure of DNA/RNA and proteins using simple analogies and visual aids.
00:10:00 - 00:15:00
The speaker explains bacteria's ongoing battle with viruses, introducing bacteriophages and their method of injecting DNA into bacteria. This sets the stage for CRISPR's role as an adaptive immune response to these viral threats, evolving over years to protect bacteria from these invasions.
00:15:00 - 00:20:00
CRISPR is described as part of a bacterial adaptive immune system, recognizing specific viral DNA sequences and incorporating them into its genome to defend against future attacks. The speaker introduces 'CRISPR' as a repeat sequence pattern in DNA, likening it to a memory of past viral invasions that helps bacteria cut viral DNA accurately.
00:20:00 - 00:25:00
The Cas protein's function in the CRISPR system is highlighted, showcasing a practical demonstration with pool noodles to illustrate DNA interaction. This part of the system processes viral DNA into 'spacers' within the CRISPR array, preparing the bacteria to recognize and destroy similar viral sequences in the future.
00:25:00 - 00:30:00
The presentation details the formation of pre-CRISPR RNAs from the CRISPR array and explains how these RNAs are used to recognize and cut invading viral DNA. Adaptations enabling bacteria to prevent self-destruction by utilizing specific recognition sequences on viral DNA are discussed.
00:30:00 - 00:35:00
The discussion shifts to the applications of CRISPR, particularly in gene editing and disease treatment, using examples like sickle cell anemia and cystic fibrosis. The potential of CRISPR to modify or correct mutations in human genes is emphasized, noting the advanced diagnostic capabilities it provides.
00:35:00 - 00:40:00
The speaker explores CRISPR's possible applications in non-human contexts, such as altering mosquito genes to combat malaria. They also discuss potential uses in agriculture and improvements in modifying genes for beneficial traits in livestock.
00:40:00 - 00:45:00
Challenges with CRISPR implementation are acknowledged, focusing on delivery complexities in multicellular organisms and the potential for unintended genetic variations. The difficulties in using CRISPR as a precise gene-editing tool, particularly when applied to complex eukaryotic systems, are highlighted.
00:45:00 - 00:50:00
Ethical considerations in CRISPR use are discussed, particularly in germline editing, which could lead to designer traits in humans. The ethical implications of CRISPR's power to alleviate or potentially misuse this technology are debated, with a call for careful regulation and international cooperation.
00:50:00 - 00:58:34
Concluding the talk, the speaker highlights CRISPR's dual essence as both a powerful diagnostic tool and a means of potential genetic modification. They encourage continued dialogue on the ethical use of CRISPR to prevent misuse and promote its benefits in medical and agricultural fields.

Mind Map

Video-Fragen und Antworten

What is CRISPR?
CRISPR stands for "Clustered Regularly Interspaced Short Palindromic Repeats," a system in bacteria that acts as an adaptive immune mechanism by identifying and cutting foreign DNA.
How does the CRISPR-Cas9 system work?
The CRISPR-Cas9 system uses a guide RNA to identify and bind a specific DNA sequence, where the Cas9 protein then makes a cut, effectively disabling the targeted DNA sequence.
What are some potential applications of CRISPR?
CRISPR can be used for gene editing to correct mutations, develop treatments for genetic diseases such as sickle cell anemia and cystic fibrosis, and even modify organisms like mosquitoes to control disease spread.
What are the ethical issues surrounding CRISPR?
Ethical issues include the potential for designer babies, accessibility, control over gene editing rights, and the implications of modifying human embryos.
What are the limitations of CRISPR technology?
Limitations include the challenges of delivering CRISPR into human cells, potential off-target effects, and the unpredictability of DNA repair post-editing.
What is the significance of the PAM sequence in CRISPR?
The PAM (Protospacer Adjacent Motif) sequence is crucial for identifying and cutting the correct DNA sequence, preventing the system from cutting its own DNA.
Can CRISPR be used in cancer treatment?
CRISPR holds potential for cancer treatment by potentially correcting mutations in tumor suppressor genes, though research is still ongoing.
What role does Cas9 play in the CRISPR system?
Cas9 is a protein that binds to the guide RNA, recognizing and cutting specific DNA sequences as dictated by the CRISPR process.
Can CRISPR be used to create vaccines?
While CRISPR's use in direct vaccine creation is uncertain, it can potentially be used to alter immune responses or identify viral components for diagnostics.
How does CRISPR relate to epigenetics?
CRISPR can potentially be used to demethylate DNA regions, affecting gene expression, although this application is still under exploration.

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Untertitel

Automatisches Blättern:

00:00:00
(applause)
00:00:02
>> Yeah.
00:00:04
All right.
00:00:06
Well, thanks for coming.
00:00:08
Some of my students are here, and that's just voluntary,
00:00:11
which is amazing.
00:00:12
So...
00:00:15
So I wanna thank Tim for actually putting these together.
00:00:22
You know, you get used to kinda doing the same thing
00:00:24
over and over again.
00:00:25
And you-- and I think it's neat to actually have something
00:00:29
to talk about that-- I'm not a CRISPR scientist.
00:00:32
I will tell you a little bit about what I do.
00:00:35
But reading about it over the last past year
00:00:37
and doing honors projects with students
00:00:40
has really given me an opportunity
00:00:42
to just kind of expand my mind,
00:00:44
which, without people like Tim pushing us constantly,
00:00:48
like, almost annoyingly... (laughing)
00:00:52
even when I was sitting up here,
00:00:54
he was trying to get another talk next year.
00:00:55
So, but anyways, I think it's good.
00:00:59
I think it's good for all of us to be thinking
00:01:01
about new things.
00:01:02
So first of all, why am I interested in this topic?
00:01:07
Well, when I was in graduate school,
00:01:09
one of the things I worked on was
00:01:10
I did work on DNA arrays for different organisms.
00:01:14
And I looked at recognition for DNA.
00:01:17
And one of the things that we'll talk about today
00:01:19
with CRISPR is that one of the things
00:01:21
that makes it unique is it identifies
00:01:23
unique sequences of DNA.
00:01:25
And so, I have a past background of doing amplification
00:01:29
and recognition of big biomolecules,
00:01:32
and then trying to analyze them in the gas phase.
00:01:35
So we were doing more diagnostic components with DNA.
00:01:39
I've worked a little bit with some students
00:01:43
and faculty out at Hope looking at doing mutations
00:01:46
in genes and seeing how those ultimately affect an organism,
00:01:52
and so, protein modifications.
00:01:54
And then, I have a couple students here
00:01:56
that did honors projects in reading this book.
00:02:00
I found this a couple years ago--
00:02:01
it's "A Crack in Creation."
00:02:03
I know a few of you have read this,
00:02:05
and it is about scientist Jennifer Doudna
00:02:08
out at Berkeley that helped develop this tool.
00:02:13
And she's an RNA chemist.
00:02:15
And so, it's not like she knows bacteria
00:02:17
or different things.
00:02:18
She looks at structures of RNA molecules.
00:02:21
So, and I just think it's kind of an interesting story.
00:02:25
So if you read what I wanted to cover today,
00:02:29
it was really looking at how this process came about,
00:02:32
and then talking about, like,
00:02:33
"What are some of the applications?"
00:02:36
And so, you can ask me questions,
00:02:38
you can comment at the end.
00:02:40
Maybe you know something I don't know, I hope.
00:02:42
Or you can say, like, that was the most ridiculous thing.
00:02:45
This is actually true.
00:02:47
So-- but I'm gonna kinda take you through this process.
00:02:51
And you-- this has been showing up in our news
00:02:56
over and over again, this concept of gene modification
00:02:59
and CRISPR.
00:03:00
And some of the things that goes on in science
00:03:04
is when a new technique is developed,
00:03:07
there are patent disputes.
00:03:08
And so, you look at some of the high-power researchers
00:03:12
in CRISPR out at Berkeley or MIT,
00:03:15
and they fight over the uses of these things.
00:03:17
And it's because these have strong diagnostic tools.
00:03:21
That means money's gonna probably be made.
00:03:24
And so, modifications around this.
00:03:26
So people are kinda vying for patents around this.
00:03:29
So you see it in the news around patents.
00:03:32
If you were at the end of the year last year,
00:03:34
you might've seen that a scientist in China
00:03:38
had modified some embryos so that they were HIV-resistant.
00:03:45
And so, I'll talk about that a little bit.
00:03:47
That's a little bit controversial.
00:03:50
And then, like I said, with patents come stocks.
00:03:54
And you will-- I know some people that have owned these,
00:03:57
and it's an emotional roller coaster
00:03:59
if you own any stock around CRISPR,
00:04:02
because it goes up and down with FDA approvals.
00:04:05
And so, we'll talk about some of the limitations of it, too.
00:04:08
And I'm not gonna give you stock tips, though.
00:04:12
But what I'd like-- so the outline
00:04:14
for my presentation is I am gonna give you
00:04:16
a quick overview of biochemistry.
00:04:19
And it's just so that when I talk about this system
00:04:22
and I talk about nucleic acids or amino acids,
00:04:26
you know what I'm-- you can at least reference back to this.
00:04:29
And if you do wanna copy this,
00:04:30
I'm happy to send this to you,
00:04:31
'cause I know probably in your pastime,
00:04:33
that's what you wanna review, is this talk.
00:04:36
(chuckling) But I will say
00:04:38
that I have sat through many of these.
00:04:40
My hope is that you take like one or two things home with you.
00:04:44
And I'll try and be short,
00:04:46
'cause everybody loves when these get out early,
00:04:48
and you get a cookie, it's great.
00:04:50
So I am gonna talk a little bit about bacteria.
00:04:53
I'm not a biologist, but-- and bacteriophage.
00:04:57
A few definitions, because I think
00:04:59
you can get caught up in the terminology.
00:05:02
Adaptive immune system, I'll explain that
00:05:04
when we get to it.
00:05:05
And then, what are some of these applications
00:05:07
of this CRISPR-Cas9 system?
00:05:10
And it's fascinating.
00:05:14
And I am gonna talk about a lot of the limitations, too,
00:05:17
because I don't-- I--
00:05:20
it's like any drug delivery.
00:05:21
Getting it to cells is tricky.
00:05:23
And so, we'll chat about that.
00:05:25
So you know, if you've taken a general biology class
00:05:28
or a chemistry class before, that DNA--
00:05:31
if you ask even a little kid what DNA is,
00:05:34
they'll say it's what makes us "us," right?
00:05:37
And we know that from a genetic perspective,
00:05:41
DNA codes for genes, right, and those genes
00:05:44
can be transcribed to RNA,
00:05:46
which is a compound that can ultimately be used
00:05:49
and translated into proteins, okay?
00:05:52
And proteins are what carry oxygen around our blood,
00:05:56
they break down hydrogen peroxide in our body.
00:05:59
All the systems that you've learned
00:06:01
around glycolysis and the Krebs cycle,
00:06:03
they all have a component in them.
00:06:05
So I'm gonna talk about DNA and RNA and proteins,
00:06:08
'cause they're all involved in this process, okay?
00:06:12
So nucleic acids, that's where we're gonna start.
00:06:15
And we have to have some molecules up here,
00:06:16
because when you think of DNA,
00:06:19
that's what you're used to, right?
00:06:20
Seeing the double helix and the kind of two rungs of the DNA.
00:06:25
But you know that it is made up of those four bases--
00:06:27
adenine, thymine, guanine, and cytosine.
00:06:31
And they pair up really specifically.
00:06:33
And that's sort of the crux of this technique
00:06:36
is something has to make sure that they pair up,
00:06:38
when DNA pairs up with each other.
00:06:40
When we recognize DNA,
00:06:42
it's because of those As and Ts and Cs and Gs, okay?
00:06:47
Nucleic acids can be in the form of RNA, as well.
00:06:50
So DNA gets coded to RNA.
00:06:52
And usually we think of this as single-stranded.
00:06:56
But it's way more complex than that.
00:06:58
The RNA folds up into proteins-- or excuse me,
00:07:00
folds up into structures like this one right here,
00:07:03
which certainly looks like it's paired up with one another.
00:07:07
And it's why Professor Doudna actually got involved in this,
00:07:11
because there is a lot of RNA kind of super--
00:07:14
that secondary-type structure there, okay?
00:07:17
So nucleic acids, RNA, and DNA.
00:07:21
And then, when you think about proteins,
00:07:23
and I'm gonna stop with the biochemistry here in a minute,
00:07:27
amino acid chains are proteins.
00:07:29
So that DNA codes for RNA, which codes for a protein.
00:07:33
And proteins are made up of these simple little
00:07:36
amino acids, long chains of them.
00:07:38
In fact, the Cas9 system, the gene editing tool
00:07:42
that I'm gonna talk about,
00:07:43
actually has about a little over 1,300 of these,
00:07:47
so 1,300 of these strung in a row.
00:07:51
And so, that gets untenable from a chemicals perspective.
00:07:56
It's hard to, say, show you 1,300 times 10
00:08:02
or 130,000, 140,000 atoms.
00:08:05
So we use cartoons.
00:08:07
And you've probably seen those before,
00:08:09
kinda those alpha helices
00:08:11
and those sheets, those beta sheets.
00:08:13
And the structures that I'm gonna show you
00:08:15
are gonna be more like cartoons
00:08:17
than they are gonna be like, sadly,
00:08:19
than like atoms strung together, okay?
00:08:23
Again, it's why people love biology,
00:08:25
and then they come to chemistry
00:08:27
and they see this, and they're like,
00:08:28
"Can we do this again?"
00:08:30
So here's a couple protein structures.
00:08:34
And you know they're cartoons!
00:08:35
They're cartoons of those alpha helices
00:08:38
and those beta sheets.
00:08:39
This is hemoglobin.
00:08:40
It's kind of a low-resolution picture.
00:08:42
This is a beautiful complex between a protein
00:08:45
and a piece of DNA.
00:08:47
And you can see the characteristic double helix
00:08:50
right across the top, and then the protein over it.
00:08:53
And I'll come back to that structure again here.
00:08:55
I'll actually come back to both of these.
00:08:58
So there is-- so we know we have our biomolecules,
00:09:03
our big biomolecules of RNA and DNA and proteins.
00:09:07
But this story sort of begins with bacteria.
00:09:09
And over 20 years ago, I thought this
00:09:12
was an interesting statement about bacteria.
00:09:15
It says they don't have easy lives, right?
00:09:17
So we eat them, and we break them down in our immune system,
00:09:22
and I read that half the bacteria die every day,
00:09:27
every couple days.
00:09:28
And I've talked to a few of you about that.
00:09:30
I'm not sure it's true.
00:09:31
But I like the idea of it, that they're under attack.
00:09:35
The point isn't whether or not that's statistically correct,
00:09:38
'cause some are growing faster and some are growing slower.
00:09:40
The fact is bacteria are under attack, right?
00:09:44
They're under attack by our guts,
00:09:47
as well as these viruses that exist
00:09:49
just to break them apart and use them, really.
00:09:54
So when you think about a virus, a bacteriophage,
00:09:59
they land on the surface of a bacteria.
00:10:01
And they kinda look like that cool land rover, right?
00:10:04
Right here.
00:10:05
And here's a nice little structure of them.
00:10:08
And they-- if you know anything about a virus,
00:10:11
they insert their DNA into that bacteria.
00:10:14
And then, they use it to make more of themselves, right?
00:10:18
And then, they lyse open and take advantage of it.
00:10:20
So you know that the cell fate when a virus is on it
00:10:24
is going to be death, right?
00:10:26
It's going to use it, it's gonna burst it open,
00:10:29
and spew out a bunch of itself again.
00:10:32
And so, what has happened over years of evolution
00:10:35
is it's developed a system to combat these things
00:10:39
when they come onto their surface
00:10:41
and inject their DNA.
00:10:43
And that's where CRISPR comes in.
00:10:45
What I like about this is it was over 20 years ago
00:10:48
when it was first identified, this CRISPR system.
00:10:51
And so, I have-- I'm gonna use pool noodles to do this,
00:10:55
because it's a visual.
00:10:56
And that's the only way I can really think about this system.
00:10:58
And Tim's gonna help me here in a minute.
00:11:01
But if you think about this as a piece of DNA--
00:11:04
and it's a cartoon, obviously, here.
00:11:06
But if you think of these as kind of genes
00:11:08
and these little segments where you can see there's purple
00:11:11
and a color and purple and a color and purple.
00:11:14
And in science, you know that we look for patterns, right?
00:11:17
And so, back in 1987, it was identified
00:11:21
that this pattern was here.
00:11:23
But then, it took 20 years to figure out
00:11:25
that it is actually part of an adaptive immune system, okay?
00:11:29
The adaptive immune system of these bacteria.
00:11:33
And so, we'll get into how this actually happens here.
00:11:37
But when you think of CRISPR, I never--
00:11:39
I was a LASER chemist
00:11:41
and I always forgot what "LASER" stood for.
00:11:44
You know, it was "Light Amplification
00:11:45
"of Stimulated Emission of Radiation."
00:11:47
You could never-- I never remembered that.
00:11:49
I never remember what CRISPR stands for.
00:11:51
But the words "Clustered," right?
00:11:54
Close together.
00:11:55
"Regularly" means they have a specific size
00:11:59
associated with them.
00:12:00
"Interspaced, Short--" not long, and that's relative.
00:12:05
I'll show you that.
00:12:06
And then, a "Palindrome" sequence in biology
00:12:10
and chemistry matters.
00:12:12
So it's the same forwards and backwards.
00:12:14
And what that does is it allows DNA or RNA
00:12:17
to have a specific sequence.
00:12:19
They'll pair with themselves
00:12:21
and make hairpin structures because of it.
00:12:23
And I'll show you a few of those.
00:12:24
And then, "Repeats," over and over and over again.
00:12:27
And I hope someone at the end asks me
00:12:30
how many times does it repeat over and over again,
00:12:32
because I'm ready for that one.
00:12:34
So we're gonna chat a little bit about what these are.
00:12:38
So that's what CRISPR stands for.
00:12:40
That basically is recognizing this pattern down here,
00:12:44
this repeated "repeat spacer, repeat spacer" sequence.
00:12:49
Now, when we talk about Cas genes--
00:12:52
so this is kind of a hybrid,
00:12:54
this system that I'm gonna talk about is a system hybrid
00:12:58
between proteins and nucleic acids,
00:13:01
so DNA, RNA, and proteins.
00:13:05
So something has to play the role of these genes,
00:13:09
the proteins, everything when they interact
00:13:11
with viral DNA or the DNA within the bacteria,
00:13:16
it's usually a protein with it.
00:13:17
So I'm gonna play that role as the protein.
00:13:20
I actually specifically wore "Cas" here
00:13:23
so that you can see this, the Cas protein.
00:13:25
I thought to myself this morning
00:13:27
that I ruined a perfectly good t-shirt,
00:13:29
unless someone goes-- 'cause I'll wear it,
00:13:31
and someone will go, "They added a C and forgot an S,"
00:13:34
you know, when-- (audience laughing)
00:13:35
so, but I'm going to-- I'll be--
00:13:38
whenever we're talking about proteins,
00:13:39
I'm gonna play that role.
00:13:41
I'm gonna be that amino acid chain.
00:13:44
And these Cas proteins do lots of different things.
00:13:47
So they are nucleases, which means they cut up DNA.
00:13:51
They separate, because if you're gonna deal
00:13:53
with viral DNA that's coming in,
00:13:55
you have to be able to separate it apart.
00:13:57
And then, they do other things,
00:13:59
like identify unique sequences, as well.
00:14:03
So whenever I'm holding the DNA, remember, I'm the protein, okay?
00:14:08
So let's get into what this means.
00:14:10
So I'm a bacteria.
00:14:13
Let's say the stage is the bacteria up here.
00:14:15
And I'm gonna be invaded by a particular virus.
00:14:18
And Tim's gonna be that virus.
00:14:21
He's gonna be carrying the DNA in here.
00:14:23
And there are basically three fundamental stages
00:14:27
of this bacterial adaptive immune system.
00:14:30
So they wanna get ready so that when a virus attacks them,
00:14:35
they know what they're going to do.
00:14:36
They'd like to destroy that viral DNA
00:14:38
before it actually takes over and destroys that bacteria.
00:14:44
So the first stage of this adaptive immune system
00:14:49
is actually taking a piece of this DNA
00:14:53
out of this virus that's coming up.
00:14:56
So Tim, if you'd come up?
00:14:58
What happens is when he brings-- oh, he already did it.
00:15:02
Yeah. (chuckling)
00:15:03
So-- yeah, and it happens-- there's a mechanism--
00:15:08
that's all you have to do-- I appreciate it.
00:15:10
I'm gonna call you back up for more.
00:15:11
(audience laughing)
00:15:12
You did your job.
00:15:13
So I, as a Cas protein inside of this bacteria,
00:15:19
I am-- like Cas1 and 2,
00:15:21
I'm gonna take this, when it gets inserted
00:15:23
into the bacteria through the cell membrane.
00:15:26
And I'm gonna look for the specific spot on this.
00:15:30
And you'll notice that I have this black line here.
00:15:32
I'll explain what it is.
00:15:33
When I recognize that, I'm actually gonna clip out
00:15:36
a little piece of DNA from that viral DNA, okay?
00:15:40
And then, that obviously gets cut up and it's gone.
00:15:44
And I'm gonna insert it on the end of my CRISPR array.
00:15:48
So remember, this is the normal bacterial genes
00:15:51
that code for, like, me, the Cas gene,
00:15:54
the things that are involved in this system.
00:15:56
And then, you'll notice it's been exposed
00:15:57
to a couple viruses.
00:15:59
And if you don't mind holding those up.
00:16:01
Like, those might be DNA from other viruses,
00:16:03
like a red piece, a little turquoise-y piece,
00:16:07
and now this yellow piece that I'm gonna add to this, okay?
00:16:11
So I've taken a piece of DNA from a virus
00:16:14
and incorporated it into my own DNA.
00:16:17
I'm a bacteria, right?
00:16:19
So now, I have this CRISPR system that has--
00:16:22
I've just added a new repeat to it.
00:16:24
So it's got this repeated pattern, a spacer...
00:16:28
that's what type of DNA now?
00:16:32
>> (indistinct). >> Viral DNA.
00:16:34
These different viral DNA.
00:16:35
I've actually incorporated it into my own DNA.
00:16:39
So it matches the sequence of other viruses, right?
00:16:44
So this yellow, if I am exposed
00:16:46
to another yellow piece of DNA, I have a matching sequence.
00:16:51
So I have these genes and these repeated sequences.
00:16:55
They're not very long.
00:16:57
And this actually is kind of a key to how this system works.
00:17:00
You notice they're about 20 nucleotides long.
00:17:03
And that's gonna come into play.
00:17:05
I'm gonna talk about that here again later,
00:17:08
because it has to do with statistics.
00:17:11
The longer the piece you come in,
00:17:13
the more specific it would be,
00:17:14
because, you know, if everything has an A,
00:17:19
then an A and a T, there's less likely,
00:17:22
and then A, T, C, statistically it becomes less likely
00:17:26
to come across a longer pattern.
00:17:30
So they incorporate this 20--
00:17:31
about 20 nucleotides, and then, this other little piece of DNA
00:17:35
that they use over and over again,
00:17:37
that black piece, that's about 30 nucleotides.
00:17:40
So a total of 50 in this CRISPR array, okay?
00:17:46
You'll notice it says Cas1 and 2 there.
00:17:50
This is the Cas1 and 2 system.
00:17:53
So bacteria have this gene,
00:17:57
like those Cas genes make this particular protein.
00:18:00
And it's pretty complex.
00:18:01
It's got, like, lots of amino acids in it.
00:18:05
It's a pretty big protein.
00:18:07
But the distance between it actually determines--
00:18:10
that's what I think is kinda elegant,
00:18:11
it keeps cutting the exact same size
00:18:14
of piece of DNA because of its length,
00:18:18
the protein length of it.
00:18:19
So it binds to viral DNA.
00:18:22
This would be the virus.
00:18:23
And it clips that little piece of DNA
00:18:26
and adds it to its CRISPR sequence, okay?
00:18:30
So DNA, according to our central dogma,
00:18:35
becomes RNA, right?
00:18:37
So DNA codes for RNA.
00:18:39
So what the bacteria does is it turns this sequence now
00:18:43
into little pieces of RNA.
00:18:45
And this is called "pre-CRISPR RNA."
00:18:48
And you'll notice it has different colors,
00:18:50
and it has that little hairpin sequence, as well, okay?
00:18:55
So what it does is it takes this off,
00:18:58
an enzyme would come along and make this piece of RNA, okay?
00:19:05
And then, it would break it up into little pieces
00:19:09
like this, so it expresses these.
00:19:11
And then, there'd need to be more of me.
00:19:14
But I would hold these in my hand.
00:19:16
So I am ready for these viruses to come in-- into the--
00:19:19
so I'm gonna ask Tim to come up with a new one here.
00:19:24
So if you think of this system now,
00:19:26
I have this-- I'm a protein with this one,
00:19:31
I'm a protein with this one, or I'm a protein with this one.
00:19:33
It looks like he's coming in with some red here.
00:19:36
So what I'm gonna do with this Cas9 system
00:19:41
is I'm gonna come up and I'm gonna go,
00:19:43
"All right, this matches, right?"
00:19:46
I'm gonna look for that black line.
00:19:48
It matches.
00:19:49
And I'm gonna cut it.
00:19:52
What happens to the viral DNA when it's cut?
00:19:56
It doesn't work anymore!
00:19:57
So now, that virus is no good anymore.
00:20:00
So I'm gonna bring the other one up.
00:20:02
I am this Cas9 system with this piece of DNA.
00:20:06
I come up, I look for this sequence,
00:20:08
I match it up, and then I cut it.
00:20:11
And now, that virus is no good anymore.
00:20:13
So I am just, like, a virus-destroying machine,
00:20:17
as long as I've seen it already, right?
00:20:20
So if I've seen it, I've incorporated it into my DNA,
00:20:24
I make the RNA from it, and then I have this protein
00:20:28
that, when this virus inserts a piece of DNA,
00:20:33
this is the Cas9 system.
00:20:35
It recognizes it.
00:20:37
This PAM sequence is that little mark
00:20:39
that I'm gonna talk about.
00:20:40
And I bind to it, I make sure that it matches,
00:20:43
and then I cut it.
00:20:45
And when you cut viral DNA,
00:20:46
you make it so that it can't use the machinery
00:20:49
in the bacteria to make more of itself anymore.
00:20:53
So that's all the CRISPR system is doing.
00:20:55
It's taking, incorporating a short piece of DNA
00:20:58
from a virus into its own genome,
00:21:01
it's turning it into RNA, and turning it into a system
00:21:05
that recognizes DNA in foreign, invading bodies, okay?
00:21:11
So I keep talking-- and you'll notice--
00:21:15
this is actually an important thing.
00:21:17
So when you leave and if you ever wanna read about this
00:21:19
before, you'll notice there's some limitations
00:21:22
to this system, is there always has to be
00:21:25
this little black mark.
00:21:27
And this is usually a three-nucleotide sequence
00:21:30
that me, as a Cas enzyme, has to recognize before I'll cut.
00:21:36
Now, this is actually pretty important,
00:21:38
because if you'll notice--
00:21:40
and it doesn't matter the order of this.
00:21:46
This worked so well in my mind. (laughing)
00:21:49
So if you look at this sequence here...
00:21:54
Let's put that one on.
00:21:55
Oh, that's the problem.
00:21:56
Hang on.
00:21:58
We can edit this out.
00:22:00
If you think of this sequence here,
00:22:03
the difference between it is this piece of viral DNA
00:22:10
has that black line on it.
00:22:12
My DNA inside the bacteria
00:22:15
does not have that black line.
00:22:17
And so, you've heard of autoimmune diseases
00:22:20
where your own immune system attacks, right,
00:22:23
your own cells.
00:22:24
What this little sequence--
00:22:25
it's called the "protospacer adjacent motif"--
00:22:29
and it's three nucleotides that the Cas gene-- me--
00:22:33
looks for before I'll cut.
00:22:36
So I would go along my own DNA as a bacteria,
00:22:39
and I'd go, "Oh, my gosh, that sequence looks like
00:22:41
"what I wanna cut."
00:22:42
But it won't, because it doesn't have
00:22:44
that little "PAM sequence," they call it,
00:22:47
the "Protospacer Adjacent Motif."
00:22:49
So you will see this if you read about the CRISPR system.
00:22:54
That's kind of a unique thing,
00:22:56
and it ends up being a limitation,
00:22:58
because if you wanna cut DNA later,
00:23:01
you can only cut when you have a unique sequence,
00:23:03
like any random DNA, okay?
00:23:06
So it's really a self-recognition tool
00:23:09
for this system.
00:23:11
By the way, finding pool noodles in February
00:23:15
in Michigan... (chuckling)
00:23:19
it's probably like trying to buy a sled in July.
00:23:21
I think they probably are in the same warehouse...
00:23:25
especially when you want the same colors, too.
00:23:29
So they have to recognize that PAM sequence
00:23:32
before they'll cut, okay?
00:23:35
So back to-- so that's virally,
00:23:38
and from a bacterial perspective, what happens.
00:23:41
Bacteria clip out a little piece of DNA, save it.
00:23:45
When they see something just like it, they cut it, okay?
00:23:49
Where this system starts to get interesting
00:23:51
is in order for this Cas9 system,
00:23:56
this protein to function,
00:23:57
it needs CRISPR RNA.
00:23:59
So it needs this.
00:24:04
It needs a sequence that matches a virus, or anything,
00:24:09
this kind of random spacer that forms that loop,
00:24:13
and then it also needs another piece of RNA
00:24:16
that they don't really know exactly what it does,
00:24:19
but they know that it helps prop up the protein,
00:24:22
like, it's a structural thing.
00:24:24
And I'm gonna show you an image of it here.
00:24:26
This is an image I found from that Doudna lab
00:24:29
where it's this Cas9 system.
00:24:32
And what Berkeley is famous for
00:24:35
is they took and made this into one piece of RNA.
00:24:39
So instead of needing this and another piece of RNA
00:24:43
for this system to work, they linked them together.
00:24:47
And they made what's called a "guide RNA."
00:24:49
So when you think of it from an application of,
00:24:52
"How are we gonna modify some DNA,"
00:24:53
they call it a "single guide" or "sgRNA."
00:24:56
And it includes the piece that goes
00:24:59
and recognizes the virus,
00:25:00
and then this other piece that's a scaffold
00:25:03
or a structural piece of RNA
00:25:04
that seems to need to be there, okay?
00:25:07
'Cause if you take it out, it won't work.
00:25:09
So what scientists did at Berkeley
00:25:12
was they made this one piece of DNA.
00:25:14
And they have a patent on that mechanism, okay?
00:25:19
So I wanted to show you-- the protein database
00:25:22
is kind of a neat thing if you've ever done
00:25:25
any DNA or RNA or protein research.
00:25:29
I found this on their website.
00:25:32
And I just wanna show it to you,
00:25:33
'cause it gets this protein three-dimensionally.
00:25:37
And I was sort of amazed they had put some music to it,
00:25:40
which I thought was--
00:25:41
I don't know, I thought it was kinda interesting.
00:25:43
So just enjoy looking at the Cas9.
00:25:46
This is the Cas9 system, which CRISPR,
00:25:49
which we know is the array, and the Cas9 protein.
00:25:52
This is the system that goes and finds and cuts DNA.
00:25:57
So...
00:25:59
(tranquil piano music)
00:27:08
This goes on for 30 more minutes, is that okay?
00:27:10
(audience laughing)
00:27:12
But you can see, it's sort of an elegant system.
00:27:15
It's got this protein, again,
00:27:17
and you can see the different components to it.
00:27:19
It's a pretty complex protein that binds to DNA.
00:27:22
You saw the pieces of DNA coming in.
00:27:25
It cuts it, okay?
00:27:26
That's what renders the viruses inactive.
00:27:29
And that's a big deal, because if you think about this, um...
00:27:37
Oh, let me go to "So What?"
00:27:40
You have a device in a bacteria, but you could pull it out,
00:27:44
and you could say, "Well, I have this tool
00:27:47
"that can recognize sequences of DNA.
00:27:49
"20 base pairs is pretty unique, okay?
00:27:53
"And it's a snipping tool
00:27:55
"so it can recognize and cut DNA."
00:27:58
So when people say the CRISPR-Cas9 system,
00:28:00
that's what they're talking about,
00:28:02
a piece of-- well, a bacterial protein
00:28:06
that can recognize, with a piece of RNA in it,
00:28:09
that can recognize and cut other DNA.
00:28:13
So that's what we're talking about.
00:28:14
And I like that from, like, Word 2000.
00:28:20
So what happens when you cut DNA?
00:28:22
So why do we care about this?
00:28:25
So when you cut DNA, a couple things can happen
00:28:27
in eukaryotic cells.
00:28:30
If you just cut it, it sorta scrambles.
00:28:33
If there's nothing that it can match off of,
00:28:36
what happens is it will-- they call them "indels,"
00:28:39
where they have insertions and deletions into the DNA.
00:28:43
So if you have a gene that, let's say,
00:28:45
it is coding for a protein that you don't want it
00:28:47
to code for anymore.
00:28:49
If you go in with a CRISPR-Cas9 system and cut it,
00:28:53
it's gonna screw up all those bases in that DNA.
00:28:57
And it's gonna make it so the protein doesn't exist anymore,
00:28:59
the protein doesn't work anymore.
00:29:02
But even more interesting is if you put in this Cas9 system
00:29:07
to cut a piece of DNA
00:29:08
and you have a piece of DNA that's correct,
00:29:12
that has a difference in it,
00:29:14
what it will do is it'll go through
00:29:16
homologous directed repair
00:29:18
and actually make a new piece of DNA that's right or correct.
00:29:22
So it's a way of correcting problems in the DNA, okay?
00:29:27
And I'm gonna give you a couple examples of this.
00:29:30
If you've taken a biology class or you've learned
00:29:33
about sickle cell anemia, okay?
00:29:36
Sickle cell anemia, if you look over here--
00:29:38
and that was why I started with RNA to DNA.
00:29:41
This is a normal gene for DNA
00:29:45
for someone without sickle cell anemia.
00:29:47
And if you notice, it says G-A-G.
00:29:51
If you have sickle cell, you don't have G-A-G.
00:29:54
You have G-T-G, okay?
00:29:56
It's a one base pair difference.
00:29:59
So if I could design a CRISPR system
00:30:02
that recognized an area around here and cut it,
00:30:06
then I had a template that I put in with it,
00:30:09
it would do exactly what this does.
00:30:13
So it would cut it, it would find a new piece of DNA
00:30:16
with the corrected base, and it would fix it.
00:30:21
Now, I put in this slide, one of the things
00:30:23
they've tried to do is--
00:30:24
"heterozygous" means you have one correct pair
00:30:29
and one incorrect pair.
00:30:30
So they have shown that in certain cells,
00:30:34
they can cut it, and it will use the other correct allele--
00:30:38
or excuse me, the correct piece of DNA.
00:30:40
So we have two copies of DNA, right, humans do.
00:30:43
It cuts the bad one,
00:30:45
and it will use the corrected one to make it--
00:30:49
to fix it, in a sense.
00:30:50
So it'll switch that T back to an A.
00:30:53
And then, hopefully, the central dogma
00:30:55
is DNA becomes RNA becomes protein.
00:30:58
Hopefully now that protein
00:31:00
doesn't have that weird amino acid.
00:31:02
And it's not weird,
00:31:03
it just doesn't have the same charge on it.
00:31:05
And so, you get those sickle cells.
00:31:07
And it's a horrible disease.
00:31:12
So modifying DNA in a eukaryotic cell
00:31:16
would be a big thing.
00:31:17
If you can cut it, it would repair itself.
00:31:20
Another example-- and this has actually been done
00:31:22
in what's called an "organoid."
00:31:25
Tim and I have worked a little with a researcher
00:31:28
over at MSU.
00:31:29
He does some CRISPR research.
00:31:32
And I visited his lab to see how he was doing this work.
00:31:36
And they build-- they take cells
00:31:40
and they turn them into stem cells
00:31:42
and grow little organs in a Petri dish, okay?
00:31:45
And that's really neat.
00:31:48
But what they can do is, if you have cystic fibrosis,
00:31:52
you will often-- one of the most common ways
00:31:54
you have cystic fibrosis
00:31:55
is you have a three base pair deletion in your DNA.
00:31:59
So you're missing-- I don't remember what it is--
00:32:02
A-A-A, I think it is.
00:32:05
But if you're missing that, you could go in
00:32:06
and cut that region, have a corrected piece,
00:32:10
and it would fix that.
00:32:12
And it's just a teeny little deletion,
00:32:15
but it makes a huge difference.
00:32:16
If you know anybody that has CF,
00:32:19
it's based on this membrane protein
00:32:23
that helps regulate water and chloride ions
00:32:26
in and out of the cell.
00:32:28
And they get thicker mucus and it clogs those pores.
00:32:33
And a devastating disease that could--
00:32:36
if you could get a CRISPR system in
00:32:38
to cut that DNA with the correct template,
00:32:41
you could fix that.
00:32:44
This is another example of non-human,
00:32:48
but where they would have-- they have done this, actually.
00:32:52
They haven't released these, I'm sure--
00:32:54
can you imagine doing bug research?
00:32:56
Like flying bug research?
00:32:59
You know they're out, right? (laughing)
00:33:02
It would be-- I just think that would be
00:33:06
an interesting lab to visit,
00:33:07
how they contain all the mosquitoes.
00:33:10
But what they do is they do gene modifications
00:33:12
to make the females have more male characteristics.
00:33:16
And so, their sexual organs are deformed,
00:33:18
and their mouths are deformed.
00:33:20
And these are specific mosquitoes that carry malaria.
00:33:25
And so, if they release these out into the wild, right--
00:33:29
I like this-- I just like this image of mosquitoes
00:33:32
deciding they're gonna pass their genes along.
00:33:35
And they call it a "gene drive,"
00:33:39
where they have baby mosquitoes,
00:33:43
and those baby mosquitoes all have these deformed components.
00:33:47
And so, they don't pass malaria.
00:33:49
And that might not be a big deal to us,
00:33:51
because we don't live with that many mosquito-borne illnesses.
00:33:57
But in a country where there's a lot of malaria,
00:34:00
this would matter, right?
00:34:04
There are two other types of systems
00:34:06
that could potentially be used here.
00:34:08
It's called "CRISPR interference."
00:34:10
So what they do is they take this Cas9 protein,
00:34:14
and remember, it recognizes pieces of DNA.
00:34:18
And they can take this, and they can lay it on the DNA
00:34:22
and then just have it adhere there.
00:34:24
So they turn off the part of it that cuts the DNA.
00:34:27
So all they do is use it as a recognition tool.
00:34:30
And you can change the structure of it
00:34:32
to make it bind more tightly.
00:34:34
And it can go and it can interfere,
00:34:36
and basically stop genes from being expressed.
00:34:40
So you don't go from DNA to RNA anymore,
00:34:42
'cause there's this stinking Cas9 protein bonded to it.
00:34:49
So you can up-- you can upreg-- you can downregulate genes.
00:34:53
And then, they can also upregulate genes,
00:34:56
where they use the same CRISPR system,
00:34:57
they shut off the ability for it to cut,
00:35:00
but they go and they bind to a specific region
00:35:02
of DNA with other components on it.
00:35:05
So they can modify the Cas to attract things
00:35:08
that will make the gene upregulate,
00:35:10
so make more genes be created.
00:35:14
And they've done this.
00:35:15
They've done this on some organisms,
00:35:18
and they've done this on mice to increase
00:35:20
the muscle mass.
00:35:21
So they turn on the genes that make them build
00:35:23
more and more muscle.
00:35:24
So from a food distribution perspective,
00:35:27
that might be beneficial, right?
00:35:29
Your organisms create more meat,
00:35:31
if that's the type of thing you're into,
00:35:33
and you could feed more people.
00:35:39
So why aren't we seeing this, like, everywhere?
00:35:43
I think that the main component--
00:35:45
and I've read a lot about this, to how you get this into cells.
00:35:50
So think about what you have to deliver.
00:35:52
Me, a Cas protein, you have to deliver me.
00:35:55
You wouldn't wanna deliver a Cas piece of DNA,
00:35:58
'cause you don't want a bunch of me.
00:36:00
I'll keep cutting DNA.
00:36:02
So you don't want me in there.
00:36:04
So you wanna deliver a protein
00:36:06
and then that single guide piece of RNA, right?
00:36:10
So those are the two things you need
00:36:11
to recognize and cut DNA, and possibly a template, right?
00:36:15
The correct version.
00:36:17
So if I'm homozygous, I have a correct version,
00:36:21
but if I'm not, I need to deliver
00:36:23
all these things to cells to make this work.
00:36:27
So they've thought about using viruses,
00:36:29
human viruses, to deliver this.
00:36:31
But those typically deliver DNA or RNA.
00:36:35
But there is sort of some promising work
00:36:37
around these exosomes.
00:36:39
And they're basically lipid protein--
00:36:42
or excuse me, lipid membranes that,
00:36:45
in this little packet in the middle,
00:36:47
they could put a protein.
00:36:49
They could put a piece of RNA in there.
00:36:52
And then, it would kind of assemble
00:36:53
when it made it to the cell.
00:36:56
Easier said than done, though,
00:36:57
because what happens?
00:37:00
Our body mounts immune responses to these things.
00:37:03
So it's tricky.
00:37:06
When I think of this happening, like,
00:37:09
"Oh, this is gonna solve cystic fibrosis
00:37:11
"and sickle cell anemia and all the other genetic diseases,"
00:37:14
the hardest thing is getting it to the cell, okay?
00:37:18
I have read a couple promising things.
00:37:19
If you could remove the cells
00:37:21
and then put them back in, like, that has some real promise,
00:37:25
because if you can take the cells
00:37:27
out of a human, and modify them,
00:37:29
and put them back in, that's a little easier.
00:37:32
But again, you don't want Cas9 in there constantly.
00:37:37
It's just gonna continue to cut DNA over and over again.
00:37:42
So I opened with that slide on that Chinese scientist
00:37:46
that modified embryos.
00:37:49
That's pretty controversial, right?
00:37:51
Like, to modify a undeveloped organism...
00:37:59
we probably all could have an argument about that,
00:38:01
whether or not that should be done,
00:38:03
because we might say, "Well, all we're gonna use this for
00:38:06
"is to get rid of CF and sickle cell anemia."
00:38:09
But you might say, "Well, I want my offspring
00:38:12
"to have big arms or strong arms or whatever,
00:38:17
"whatever, blue eyes."
00:38:19
You might-- that might get a little more complex.
00:38:23
But you get that that's the best place to do it, right?
00:38:26
Because you've got one cell you have to modify
00:38:29
instead of-- I mean, I've read--
00:38:32
they use the analogy in this book, like,
00:38:35
"When do you edit the paper?"
00:38:37
Before it goes to print, right?
00:38:39
You edit the paper before it goes to print
00:38:41
because once you, if you modify one cell,
00:38:45
that's fantastic.
00:38:46
But once it has produced a baby,
00:38:49
it is millions and billions of cells.
00:38:52
So it's a little more difficult to do.
00:38:54
So the differences between germline cells,
00:38:58
egg and sperm, or somatic cells-- skin, organs.
00:39:02
It's a little less controversial
00:39:03
because you don't have to--
00:39:07
you know, you're not gonna pass that on to offspring,
00:39:09
but it's way more difficult.
00:39:13
There also is, if you're reading about this
00:39:17
a little bit more, DNA repair can be pretty unpredictable.
00:39:22
So it's beautiful that I showed those two slides,
00:39:25
like you get insertions and deletions,
00:39:27
and you get a correction if you have a correct template.
00:39:30
But it doesn't always work that way.
00:39:34
That Chinese scientist, what he did was...
00:39:39
there is a protein that HIV will bind to on the cells,
00:39:44
our immune cells.
00:39:45
And you know, there's a certain amount of people
00:39:49
in our population that are immune to HIV.
00:39:51
You can be exposed to it, and they have a mutation
00:39:54
on that protein in their membranes
00:39:58
so the HIV virus can't bind to it.
00:40:00
So what he did was he knocked out that P,
00:40:04
he modified that gene so that they have a modified protein.
00:40:08
Now, that's problematic because that mutation happened
00:40:12
probably millions of years ago.
00:40:14
And we have other genes-- we don't know a lot
00:40:17
about that gene that HIV connects to.
00:40:20
So if it modified just through the natural process of evolution,
00:40:26
if other genes took its place,
00:40:29
it if was important,
00:40:30
other genes actually compensate for it being mutated.
00:40:34
And so, when you just go in and blindly modify one protein
00:40:38
and you don't know a lot about it,
00:40:40
there could be other consequences for those children
00:40:44
that were born that we don't know about, okay?
00:40:47
So it is pretty controversial for a reason, right?
00:40:52
So kind of in conclusion,
00:40:55
this is an adaptive bacterial immune system.
00:40:59
Bacteria are exposed to viruses,
00:41:00
they clip out little pieces of DNA,
00:41:03
and then they create a protein-RNA system
00:41:07
that can recognize and cut DNA.
00:41:11
We know that DNA can repair itself
00:41:14
if there's a template.
00:41:15
So cutting it, there's a chance
00:41:17
that it could fix little mutations.
00:41:20
I think a lot of the interesting research
00:41:22
around binding without cutting is really important,
00:41:26
because we can upregulate genes and downregulate genes.
00:41:30
But right now, the biggest issue, I think,
00:41:33
is delivering this to cells.
00:41:35
And so, that's why egg and sperm cells,
00:41:40
pretty easy to access.
00:41:41
But if we wanted to go in and modify my lung cells
00:41:45
because I had CF, that might be a little trickier to do.
00:41:48
So that is-- I will give you a little recommendation.
00:41:53
If you wanna read a pretty good book,
00:41:55
Jennifer Doudna, again, at Berkeley,
00:41:57
RNA chemist, biologist, chemist...
00:42:01
we're all on the same team, right?
00:42:04
They, uh-- she has a--
00:42:06
it kind of takes this really good perspective on this,
00:42:09
because she looks at the power of this
00:42:12
and worries about it from an ethics standpoint.
00:42:15
And I think she's trying to get in front of it,
00:42:19
as a community, right?
00:42:21
And just because if we were to pass laws
00:42:24
or make regulations on this,
00:42:26
it's a big deal if we do it in our country.
00:42:28
Other countries could do it, right?
00:42:30
And so, science isn't limited,
00:42:32
we don't limit science based on our laws
00:42:35
in one country.
00:42:36
But she just wants to open the conversation.
00:42:39
I think she does a nice job kind of outlining this process
00:42:43
and then talking about what it could become.
00:42:48
Tim mentioned-- well, actually, Taylor mentioned
00:42:52
that sometimes we think about this
00:42:55
in terms of, like, if we were to make a designer baby
00:42:59
or something like that,
00:43:00
only people with money would be able to do something like that.
00:43:04
Where, in turn, she makes the argument
00:43:06
in her book that we maybe can't afford NOT to do this,
00:43:12
because if you could cure a disease
00:43:15
or fix something before it becomes a problem,
00:43:17
that might be cheaper in the long run.
00:43:19
And so, there are a lot of ethics around it.
00:43:21
But it's kind of a fascinating topic,
00:43:23
and I appreciate Tim for letting me come and talk about it.
00:43:26
And I'll take any questions you have.
00:43:29
So...
00:43:30
(applause)
00:43:32
I think I got done-- yeah, it's good, good timing.
00:43:37
>> I have a microphone. >> All right!
00:43:44
When I interviewed for my job 20 years ago,
00:43:47
Tom asked a pretty mean question,
00:43:50
I'm not gonna lie. (laughing)
00:43:51
>> Now, I'm gonna show my ignorance here.
00:43:53
So when a bacteria reproduces, replicates,
00:43:56
I don't know how bacteria do this,
00:43:58
does it pass on its DNA
00:44:03
that it's been changing all along here?
00:44:06
>> Yeah. >> So isn't that getting, like,
00:44:08
ungainly large after it keeps getting--
00:44:11
>> Ah, that's a good question-- it's a good way to ask this.
00:44:12
So interestingly, this is all over the place.
00:44:15
There are, like, dozens and dozens of CRISPR systems.
00:44:20
Like, there's type 1, type 2, type 3.
00:44:22
Some of them recognize RNA.
00:44:25
A typical bacteria only has--
00:44:28
that's a pretty dynamic thing.
00:44:30
So they might only have three to five CRISPR arrays.
00:44:34
I mean, they might not have many incorporated viral DNA
00:44:38
in their DNA.
00:44:40
So they pass on what they have
00:44:42
when they replicate their own DNA.
00:44:44
But I have also read that other organisms
00:44:46
can have hundreds of spacers in there, yeah.
00:44:50
So yeah, but there are limits.
00:44:52
So if you look at the rate--
00:44:54
it depends on what they're being exposed to, as well.
00:44:57
So it actually runs the other way.
00:45:00
You have the Cas genes, and they add in the middle...
00:45:03
and the things that get further down,
00:45:05
they sooner or later get clipped off
00:45:07
because they're not being used as much.
00:45:09
So it can turn it over.
00:45:11
It's not like once it's in the CRISPR array,
00:45:13
it's always there.
00:45:14
So they do clip them out after a while.
00:45:17
So, that's a good question.
00:45:20
I like that one-- I knew the answer.
00:45:21
You redeemed yourself after 20 years. (laughing)
00:45:25
>> Any other questions?
00:45:36
>> Could I ask you to comment a little further
00:45:38
on some of the ethical issues
00:45:41
that Jennifer Doudna brings up in the book?
00:45:47
I can give a specific prompt, if you like.
00:45:50
>> Oh, go ahead. >> Yeah, yeah.
00:45:51
I was curious if there's any talk
00:45:56
about who should have the right to control gene editing.
00:46:00
Of course, 'cause you'd ask,
00:46:01
do you wanna hand it off to the government?
00:46:03
Do you want patented gene sequences
00:46:05
or gene editing tools, like--
00:46:09
what is-- does she have any commentary on this?
00:46:13
>> She does.
00:46:14
Primarily what-- she does talk a lot about access to it.
00:46:19
And again, from-- it is more that--
00:46:23
so initially, she was a little horrified by it
00:46:27
because she thought of what could potentially come about
00:46:30
from this technique.
00:46:32
But then, she got to this place where she thought,
00:46:35
"If we can do this, why don't we?"
00:46:37
Because it is sort of about...
00:46:42
easing human suffering.
00:46:43
And so, she comments mostly on that.
00:46:48
She doesn't get-- obviously, there's economic
00:46:51
and different components to it.
00:46:52
And she talks about that, too.
00:46:54
But it's primarily on basically costs.
00:46:58
And she-- I mean, I think she's trying to get people
00:47:01
to talk about this from the scientific community,
00:47:04
because it is tricky.
00:47:05
Who does run it?
00:47:06
Once people have patents on this,
00:47:11
they have a patent, and so... (chuckling)
00:47:14
and I think that that's why she's kinda having the discussion.
00:47:17
'Cause right now, there are private companies
00:47:18
that own these patents.
00:47:21
I don't know if I answered your question.
00:47:23
It's pretty big.
00:47:25
>> (indistinct). >> Okay, yeah.
00:47:30
>> (indistinct). >> Yeah?
00:47:31
>> I have a question pertaining to cancer.
00:47:34
Did you-- in your readings, did you come across
00:47:37
any researchers who were trying to insert
00:47:40
the sequence that will allow cancer cells
00:47:43
to remember that they need to go into apoptosis?
00:47:48
>> Yeah, they have a lot of hopes with cancer cells.
00:47:53
In fact, over at Michigan State,
00:47:56
that's one of the interesting things,
00:47:58
is there's a common mutation in cancer cells.
00:48:01
It's a tumor suppressor gene that gets mutated.
00:48:06
And apparently, the cell type
00:48:08
that you can do CRISPR in
00:48:11
matters whether it's a cancer or non-cancer cell.
00:48:14
It's easier to do it when they're cancer cells
00:48:17
or when they have a particular mutation,
00:48:20
which makes some sense.
00:48:21
But absolutely, those are the things that,
00:48:23
if you can sequence it and find out where the errors are,
00:48:27
those are absolutely techniques that they could use.
00:48:30
Like, turn on that process again,
00:48:33
because something has been mutated
00:48:35
that it's not getting that signal to destroy itself.
00:48:38
Yeah, for sure.
00:48:45
>> So I know you brought up autoimmune diseases
00:48:48
at the beginning of the talk,
00:48:50
but I just wanted to ask you to expand on that a little bit.
00:48:53
What are the hope-- what's the hope
00:48:56
with CRISPR technology in the treatment
00:48:59
of autoimmune diseases,
00:49:00
and is there actually any scientific evidence
00:49:03
that that could come about?
00:49:05
>> Yeah, that I actually don't know.
00:49:06
I will tell you, I was making the analogy
00:49:09
that the reason they have this little--
00:49:15
like, the Cas proteins actually recognize
00:49:17
this little piece right here
00:49:19
so that they don't go and cut their own DNA up.
00:49:22
So they don't-- the bacteria don't really have,
00:49:25
in a sense, an autoimmune disease
00:49:26
where they'll cut, 'cause the bacteria
00:49:28
has no interest in cutting itself up.
00:49:31
So it has a mechanism where the piece
00:49:34
it nicks out of a virus,
00:49:36
it has to be next to a specific sequence.
00:49:39
And the most common one is N,
00:49:41
which just means any amino acid--
00:49:43
or excuse me, any nucleic acid, G-G.
00:49:46
And so, it'll search along here, see A-G-G,
00:49:50
and then clip out this piece to incorporate into its DNA.
00:49:53
But it doesn't put N-G-G into its DNA,
00:49:56
because it would go and cut itself then.
00:49:59
And so, it was more of a-- I haven't read much
00:50:03
about autoimmune diseases in CRISPR, though.
00:50:07
I mean, autoimmune diseases are such--
00:50:09
are typically where you have a component
00:50:14
that's self-recognizing.
00:50:15
So I guess, could you go and cleave out
00:50:18
that section in your immune system?
00:50:21
Boy, potentially.
00:50:23
But I'm sorry, I don't know that much about it.
00:50:27
Sounds like you have a project now. (chuckling)
00:50:31
Other questions?
00:50:35
Oh.
00:50:39
>> I was wondering-- it's kind of a two-parter--
00:50:41
is CRISPR interference
00:50:43
preferred in any way over using an siRNA or microRNAs,
00:50:48
or is that like a quality check step
00:50:52
before proceeding on, like seeing a downregulation
00:50:55
in the protein before actually trying to modify it?
00:50:58
>> Yeah, I think, that's pretty specific.
00:51:02
I know RNA interference is--
00:51:06
I would think that this could have a higher affinity
00:51:08
for a piece of DNA, 'cause you know that any bacteria--
00:51:13
or excuse me, any protein,
00:51:14
you can do kinetic studies on it
00:51:16
where you modify it and see how strongly
00:51:19
it bonds to a segment of DNA.
00:51:21
So I would guess that CRISPR would give you kind of a control
00:51:25
that just throwing another piece of RNA in it
00:51:27
to interfere wouldn't provide, right?
00:51:30
So I would think that this would be a preferred mechanism.
00:51:33
But I know there are definitely therapeutic treatments
00:51:38
where they just are putting RNA to interfere with proteins
00:51:42
being created, so I would think this would be preferred.
00:51:47
Does that answer your question?
00:51:48
Was there another part of it?
00:51:54
>> Any other questions? >> Oh, Taylor.
00:51:59
>> Do you know if there's been any research in using CRISPR
00:52:01
to develop vaccines or anything like that,
00:52:03
as far as altering the immune system to be preventative
00:52:06
in seeking out certain viruses that we can't right now?
00:52:10
>> (forceful exhale) I don't.
00:52:11
I will say that I did read a few articles
00:52:15
where viruses will put latent bacteria in our bodies, right?
00:52:20
They will insert-- and what they have done is,
00:52:25
with CRISPR, you can identify really low concentrations
00:52:28
of things.
00:52:29
So I know from a diagnostic standpoint,
00:52:31
that's where I think a lot of this future is,
00:52:34
is in diagnostics.
00:52:35
So like, if you had a virus still in your system
00:52:40
but you weren't showing any symptoms,
00:52:43
could you actually go and clip that out
00:52:45
or identify that it's even still there?
00:52:47
But in terms-- the immune system's pretty complex.
00:52:51
(chuckling) I'm not an immunologist, so...
00:52:55
nor do I wanna give a talk on it next year, Tim.
00:52:58
(all laughing)
00:53:01
Anything else?
00:53:08
>> So Bill, there's a lot of controversy
00:53:10
about where this should or shouldn't be used.
00:53:12
Where do you think, or where do scientists in general think,
00:53:16
are the least controversial uses for CRISPR?
00:53:19
Where will we see this come along first?
00:53:22
>> Yeah, so some of that work that I read on--
00:53:27
with exosomes is I do think that--
00:53:31
so ex-- so cells apparently put out--
00:53:35
they communicate with these components
00:53:38
that will have proteins and DNA in them.
00:53:43
I would think that they could target specific organs
00:53:45
or disease using this system, as opposed to like--
00:53:49
which wouldn't be that controversial, right?
00:53:52
I think-- what I will tell you,
00:53:55
the more I read about this,
00:53:56
the more I see it as a diagnostic tool
00:53:59
as opposed to a tool that's gonna go in
00:54:01
and modify people's DNA,
00:54:03
unless it's on germline cells, truthfully.
00:54:07
And it's because it's pretty unpredictable
00:54:10
where this Cas protein will, one,
00:54:13
how long it'll stay in a system,
00:54:15
and when as long as it's there,
00:54:16
it's cutting DNA.
00:54:18
And they get off-site cuts.
00:54:21
It's not as specific as it's being touted as.
00:54:24
So... I think that.
00:54:28
I mean, if you had-- it's like any drug.
00:54:32
I think that if you were desperate,
00:54:35
you would try it, right? (chuckling)
00:54:37
So I mean-- but I think that where you do see issues
00:54:43
and people don't like to see are germline modifications
00:54:46
that result in specific traits.
00:54:48
But using it for disease,
00:54:50
I think those are the types of things you'll see.
00:54:59
All right? >> Maybe one more question,
00:55:00
if there is any?
00:55:04
>> There might be cookies left.
00:55:05
Oh. (all laughing)
00:55:08
Yeah?
00:55:10
>> So because CRISPR keeps on cutting DNA,
00:55:13
as you've said, because it's not exactly well-regulated
00:55:17
at the moment, if it keeps on cutting DNA
00:55:20
in areas that you don't want it to,
00:55:22
couldn't that be a limitation of it,
00:55:24
where it would cause its own kind of diseases?
00:55:27
>> Absolutely.
00:55:29
So one of the things that they're doing, though,
00:55:32
is that beautiful video with it kinda looping around,
00:55:38
is there are some of those domains
00:55:40
in the Cas protein, they're modifying,
00:55:43
so they don't recognize the DNA as well.
00:55:47
So they can lower the affinity
00:55:48
that that Cas9 system has for a particular segment of DNA.
00:55:54
So it'll periodically find its target and cut.
00:56:00
But if they lower the affinity,
00:56:02
the off-site cuts don't--
00:56:04
it's sort of a give and take, right?
00:56:06
You lower the affinity for the cutting.
00:56:08
You don't cut as often.
00:56:10
But you don't hit your target as often, either.
00:56:12
And so, I think a lot of the research
00:56:14
is going to be around modifying that so it doesn't hit as much,
00:56:18
because what you don't want is to fix one problem
00:56:20
and create another problem.
00:56:23
But that's real.
00:56:28
Oh.
00:56:31
>> I lied. >> Yeah. (chuckling)
00:56:37
>> Can Cas9 bind regions of DNA
00:56:40
that are methylated more so than regions that are not?
00:56:45
>> Oh, I don't know about that.
00:56:47
I know where you're going with that, but I'm not sure.
00:56:51
I think-- I have read that they will cut out
00:56:55
areas of methylation
00:56:56
so that they don't have the effects of what that might do,
00:57:01
like turning a gene on or turning a gene off.
00:57:03
But you can get Cas systems that recognize
00:57:07
specific methylated areas, though.
00:57:10
So they're working on that.
00:57:11
I just don't know that much about it.
00:57:16
All right. >> I had a quick question.
00:57:18
When they do-- you were saying that it's easier
00:57:21
to use probably when you take the cells
00:57:23
out of the patient and modify them.
00:57:25
Is that what they're currently doing with immunotherapy?
00:57:28
Are they using the Cas9 to do that, and how come?
00:57:33
Why would that be easier to get that in those cells?
00:57:37
>> Well, I think it's because they don't
00:57:38
have to deliver it through your system,
00:57:41
like inject it into your blood or--
00:57:43
so they have it in a particular container
00:57:46
that they can-- 'cause you can change the solvents
00:57:48
around cells and make them take things up easier,
00:57:53
'cause you can put more extreme conditions on a cell
00:57:55
in a test tube than you can in your body,
00:57:58
because you have to live.
00:58:00
And so... (laughing)
00:58:01
so you don't wanna do too many extreme things
00:58:04
in a biological system.
00:58:05
But if they can pull the cells out
00:58:07
and then put them back in, like bone marrow
00:58:10
and things like that,
00:58:11
I have seen that they're doing some success.
00:58:14
But I don't know if they're doing it in humans yet.
00:58:17
But you see mouse models and things like that
00:58:19
where they are, so, yeah.
00:58:23
>> All right, well, we wanna respect--
00:58:25
>> Thanks for coming. >> Everybody's time.
00:58:26
And let's give Professor Faber
00:58:29
another round of applause here.
00:58:31
(applause)