Genome Engineering with CRISPR-Cas9

00:00:11:04 (gentle tune)
00:00:17:08 - In talking to my friends who are scientists
00:00:19:12 I think we all sort of feel that sense
00:00:21:20 of wonder that sense of discovery.
00:00:23:13 You know, this feeling that even the
00:00:25:04 even the most trivial thing that I'm doing
00:00:27:04 in the lab, I might be the first person ever to
00:00:30:06 know this little piece of information.
00:00:32:17 And it feels really interesting.
00:00:34:01 It feels really fun.
00:00:35:02 It's like, I'm, you know, I'm peeling the onion.
00:00:37:04 I'm, you know, I'm, I'm peeling back the, a little bit more
00:00:40:06 of the truth that I'm trying to learn about.
00:00:42:08 You know, how, how are, how is our world working?
00:00:49:15 - I have a lot of ideas
00:00:50:17 of different endeavors I want to pursue.
00:00:54:11 I think it's my personality a little bit
00:00:57:08 always need to be on the move.
00:01:00:08 I think as a scientist, it's good
00:01:01:17 to not always be in the comfort zone.
00:01:05:14 The first time I heard about CRISPR was around 2006
00:01:11:14 when I read some articles explaining
00:01:16:19 that CRISPR could be used as a way to classify
00:01:22:17 in a more
00:01:24:16 let's say fancy manner, different strains of bacteria.
00:01:30:02 So this was the first encountering with
00:01:33:12 with the CRISPR systems. But then I read
00:01:35:17 about it and that was it for me.
00:01:37:04 - I think I first heard about it from Jill Banfield,
00:01:39:18 my colleague here at Berkeley, sometime around 2006
00:01:44:00 and she was working on bacterial genomes and, you know
00:01:48:11 sequencing bacterial DNA, finding lots
00:01:51:02 of examples of these repetitive sequences called CRISPRs.
00:01:58:11 And hadn't probably paid a lot of attention
00:02:01:10 to them until some research that was published that showed
00:02:04:18 that those sequences included little snippets of viral DNA.
00:02:13:03 And this, you know
00:02:13:23 it was very intriguing because it suggested
00:02:17:14 that bacteria were keeping these little copies
00:02:21:06 of viral sequences around for a purpose.
00:02:25:16 Some research suggested
00:02:27:15 that CRISPRs were using these little RNA copies
00:02:31:07 of DNA sequences to detect and destroy viruses.
00:02:39:00 - I was at the time a postdoctoral fellow in the lab
00:02:42:00 of Jennifer Doudna, kind of fresh off the boat.
00:02:45:20 This was at a time when Rudolph Barrangou's Science
00:02:49:02 paper came out, establishing the CRISPR system
00:02:52:02 as kind of a primitive bacterial prokaryotic immune system.
00:02:59:17 So my
00:03:00:13 first exposure to this was, was
00:03:02:02 at a journal club we used to have in the Danner group.
00:03:07:02 I remember at the time we were, you know, speculating
00:03:09:23 how could this possibly work?
00:03:12:17 - The knowledge was really limited to at the time
00:03:14:16 what was hypothesized
00:03:16:17 as that
00:03:17:13 CRISPR was a mechanism that would allow to
00:03:20:13 recognize an infection was a virus.
00:03:24:00 And ultimately kill the viruses by targeting the genome
00:03:28:15 of the viruses using protein and RNA components.
00:03:31:20 So this was the hypothesis in 2006
00:03:37:18 - In Puerto Rico in the spring of 2011, I met Emmanuelle.
00:03:42:06 - We were given talks
00:03:44:17 at the same session, the same CRISPR session.
00:03:47:01 - She said, you know, I'd been wanting to talk to you.
00:03:50:04 I'm glad to meet you in person because I've been thinking
00:03:53:01 that you might like to collaborate
00:03:54:01 with my lab a, on a project.
00:03:57:14 We were talking about a protein that at the time was known
00:04:00:20 as csn1 and now is known as Cas9.
00:04:05:07 - It's this mysterious
00:04:06:03 csn1 protein, surely we were predicting the function
00:04:09:09 but no one could show up to now how it was really working.
00:04:15:06 - We had this idea that, okay
00:04:17:06 the Cas9 protein was somehow going to be cutting DNA
00:04:20:02 but how exactly it was going to do it,
00:04:22:06 we didn't know at the time.
00:04:25:13 - And for Jennifer Doudna it was extreme, let's say
00:04:30:14 curiosity on how really those proteins work
00:04:34:07 - At the end of the conversation,
00:04:36:21 she said to me "I think it's going to be really fun to work
00:04:39:20 with your lab on this mysterious csn1."
00:04:45:11 And I just, when she said that, "this mysterious csn1",
00:04:48:02 I just felt this little chill, you know
00:04:49:14 going down my back, thinking
00:04:50:10 you know, this, this, this really is going to
00:04:52:00 be a very interesting protein to study.
00:04:59:18 - So the collaboration ultimately went very, very fast
00:05:02:19 involved a PhD student
00:05:04:18 from my lab, Krzystof Chylinski
00:05:06:18 and a postdoc from our lab Martin Jinek.
00:05:09:20 - Our goal was to really
00:05:10:16 find out what, what the biochemical function
00:05:12:16 or the mechanism
00:05:13:13 of this Cas9 protein essentially was.
00:05:18:23 - What we do as biochemists is we set up systems
00:05:22:04 in the laboratory and ask, you know, in isolation
00:05:25:08 what does this particular molecule do?
00:05:28:18 What is its function?
00:05:30:10 - If you do the experiments with the components provider
00:05:34:12 you bring them together
00:05:35:12 in a test tube and you show that it's working
00:05:37:17 then you know that you don't need anything more.
00:05:40:07 It's self-sufficient, it's working like this.
00:05:42:09 You don't need anything else from the cell.
00:05:44:09 - And we said, well, let's see
00:05:45:16 if we can take that strategy using purified
00:05:48:18 Cas9 protein.
00:05:49:21 And we also made purified CRISPR RNA molecules
00:05:54:00 these little RNA copies of DNA sequences
00:05:57:02 and then combine them together
00:05:59:02 and then started testing to see
00:06:01:06 can it use the letters in the RNA
00:06:03:19 of that CRISPR molecule to find a unique DNA sequence
00:06:08:08 and cut it.
00:06:10:03 And initially the answer was no, no dice, nothing working.
00:06:16:15 - You know, this is a process of trial and error
00:06:20:01 - You know, and there could be some silly technical
00:06:22:08 thing going on.
00:06:23:04 Like, you know, our protein purification isn't any good
00:06:26:20 or somehow our, you know, our sample that we're testing
00:06:29:15 is just, you know, has been destroyed in some way
00:06:32:22 or maybe there's something missing from the reaction.
00:06:37:19 There was a second type of RNA that Emmanuelle's lab had
00:06:43:16 been studying as part of this CRISPR system called tracr
00:06:46:23 - tracrRNA, it was an RNA molecule
00:06:49:12 which we realized was very abundant in the bacteria.
00:06:54:22 Most likely there was an interesting function
00:06:57:23 And we realized ultimately that this RNA was encoded
00:07:01:15 by a piece of DNA that was
00:07:04:05 in the vicinity of the DNA coding for the CRISPR system.
00:07:08:05 Maybe this tracrRNA molecule has something to
00:07:11:18 see with CRISPR, maybe CRISPR needs other components.
00:07:15:00 I remember the discussions saying you need the CRISPR
00:07:21:00 proteins and the RNAs. You don't need anything else
00:07:24:18 - But we started thinking about it and wondering, you know
00:07:27:13 maybe it would be important to include the tracrRNA.
00:07:31:02 And this was actually an experiment that was first done
00:07:33:08 by Krzysztof Chylinski, the student in Emmanuelle's lab
00:07:36:07 who was our collaborator on the other side of the big pond.
00:07:40:00 And he did that experiment
00:07:41:22 and got a beautiful answer, which was that yes
00:07:45:04 when you include the tracrRNA, the CRISPR RNA
00:07:48:06 and Cas9 together in this reconstituted purified system.
00:07:53:18 Now that Cas9 protein could find a unique DNA sequence
00:07:58:15 and cut it.
00:08:00:00 - This was one of the milestones in that project, realizing
00:08:03:06 that this tracrRNA was in fact also required
00:08:06:20 for the process of cutting the DNA.
00:08:10:16 Once we knew that, then, of course, we wanted to
00:08:13:07 see what parts of the tracrRNA are needed for the process.
00:08:18:09 I did a set of experiments that suggested that certain parts
00:08:21:17 of the two RNA molecules were essentially dispensable
00:08:25:08 but they were dispensable in a way that would allow us to
00:08:29:07 fuse the two RNAs together and in doing so reduce
00:08:33:04 the complexity of the system, just go directly to DNA
00:08:37:05 cutting, without having to put these two RNAs together
00:08:39:18 Somehow. This then led to these experiments
00:08:42:16 with these, this single RNA guide fusions
00:08:45:10 - Martin had made several versions that differed just
00:08:48:13 in the letters of the sequence that would allow Cas9
00:08:52:04 to find a different piece of DNA
00:08:55:12 - By basically designing the sequence of the guide RNA
00:08:58:17 you could redirect it to essentially any sequence
00:09:02:09 in double-stranded DNA.
00:09:05:00 - So we had this thing programmed with five different RNAs
00:09:08:10 you know, separate samples that would find
00:09:10:16 in principle five different places
00:09:12:09 in the DNA and lead to a cut.
00:09:14:05 And, you know, we were, you know
00:09:15:17 in the lab looking at the result of this experiment
00:09:18:00 we're leaning over the lightbox and we could see
00:09:20:02 that each piece of DNA was a different size corresponding
00:09:25:07 to the length predicted based on the ability
00:09:28:14 of these single guide RNAs to direct Cas9
00:09:31:21 to cut the DNA at a particular place.
00:09:34:11 We had been able to engineer this thing to be different
00:09:36:23 from how it is in nature and much simpler in a sense.
00:09:39:20 - It was a, it was a great moment.
00:09:42:17 This was going to be a programmable system that you
00:09:46:17 could repurpose for other things, including genome editing
00:09:51:15 - Here was something that was true, you know, fascinating.
00:09:55:09 First of all, just fundamentally about nature.
00:09:57:18 And then thinking
00:09:58:19 about how you might be able to use that in other settings.
00:10:02:00 And other kinds of cells was, was also incredibly fun.
00:10:05:17 Cause you know, the more you thought about it
00:10:07:05 the more ideas would come cause it was just so
00:10:10:07 clearly going to be so useful for a lot of things.
00:10:13:19 So there was a very intense few weeks finishing
00:10:16:12 up the last few experiments and writing up a manuscript.
00:10:20:10 - And when we were writing the paper
00:10:22:22 actually the bright days were coming in Sweden.
00:10:27:04 It's very disturbing, physiologically speaking
00:10:29:06 because you never see darkness.
00:10:31:15 You just see like a gray sky.
00:10:34:17 I would work.
00:10:35:13 I would have a tendency to work a little bit
00:10:37:22 around the clock and to not sleep a lot
00:10:40:05 - Excitement was building up.
00:10:41:15 So the final weeks in this process, there was this
00:10:44:00 you know, playing ping pong, basically with the manuscript
00:10:47:10 - I would realize it's 3:00 AM in the morning
00:10:49:06 because I was corresponding with California
00:10:52:14 and realizing that that, no
00:10:54:12 it was really time maybe to get some hours of sleep.
00:11:02:14 And ultimately the paper that was published in 2012
00:11:06:05 in Science showing the biologists how they
00:11:09:23 could harness a system.
00:11:11:14 So providing them the guidelines or the recipes to
00:11:15:01 to use a system for their own purposes, which they did
00:11:17:10 successfully because six months after the publication
00:11:20:10 of our paper in Science, very fast we saw publications
00:11:25:22 showing that the technology was working for genome editing
00:11:30:03 and even engineering in, in plant cells, in human cells
00:11:33:17 in mice, in different model organisms, which biologists use.
00:11:37:18 So this was, this was very, very fast
00:11:39:10 actually.
00:11:40:13 - It was clearly going to be a race because if it worked
00:11:42:21 it was going to be so exciting.
00:11:45:18 I wasn't thinking about, oh my gosh.
00:11:48:10 I mean
00:11:49:11 this is a tool that, you know, it fundamentally
00:11:53:12 allows us to change our relationship with nature.
00:11:56:13 It actually allows us to change human evolution
00:12:00:12 if we want to, right? It's that, it's that profound.
00:12:05:06 And it puts into all of our hands as, as human beings
00:12:12:02 a tool that, you know, we've never had in the past
00:12:14:14 the ability to change the fundamental chemical nature
00:12:19:10 of who we are in this way, right.
00:12:20:08 And now we do,
00:12:22:15 and what do we do with that?

This Talk

Speakers: Emmanuelle Charpentier, Jennifer Doudna, Martin Jinek

Audience:

General Public
Educators of H. School / Intro Undergrad
Student
Educators of Adv. Undergrad / Grad
Researcher
Educators

Recorded: May 2017

Talk Overview

Jennifer Doudna and Emmanuelle Charpentier won the 2020 Nobel Prize in Chemistry for their development of a powerful gene editing tool known as the CRISPR-Cas9 system. In this short film, Doudna, Charpentier, and Martin Jinek, who was a post-doc at the time in Doudna’s lab, describe how their famous collaboration happened and share the story behind the influential experiments that led to their discovery.

Speaker Bio

Emmanuelle Charpentier

Emmanuelle Charpentier

Emmanuelle Charpentier received her PhD at Institut Pasteur, Paris. She served as Director at the Max Planck Institute for Infection Biology, Berlin from 2015-2018. Since 2018, she has served as the Founding, Scientific and Managing Director of the Max Planck Unit for the Science of Pathogens, Berlin. Charpentier was awarded The Nobel Prize in Chemistry… Continue Reading

Jennifer Doudna

Jennifer Doudna is a Professor of the Departments of Chemistry and of Molecular and Cell Biology at the University of California, Berkeley, and an Investigator of the Howard Hughes Medical Institute. Early in her career, she studied the structure and mechanism of ribozymes (enzymatic RNA molecules) and RNA-protein complexes. Now her research focuses on understanding… Continue Reading

Martin Jinek

Martin Jinek

Martin Jinek received his PhD in Elena Conti’s lab at EMBL. He performed postdoctoral work at UC Berkeley with Jennifer Doudna. He is now an Associate Professor of Biochemistry at the University of Zurich, where his lab studies protein-RNA interactions that regulate cellular function. Continue Reading

Credits

Sarah Goodwin (Executive Director, Science Communication Lab): Senior Producer
Steven Tyler: Editor
Gb Kim (Illustrator, Explorer’s Guide to Biology): Illustrator
Derek Reich (Zooprax Productions): Cinematographer
Eric Kornblum (Videographer, iBiology): Camera

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