Stories of CRISPR: Rodolphe Barrangou

00:00:05:11 (slow instrumental music)
00:00:16:21 - There's thousands of types of cheese in the world.
00:00:20:05 I certainly own that in my French heritage for sure.
00:00:23:07 And they rely on lactic acid bacteria.
00:00:27:23 It's a small family fellow genetically of bacteria
00:00:31:14 that transform lactose from milk into lactate or lactic acid
00:00:37:22 and acidify milk to enable coagulation.
00:00:43:16 So it turn liquid milk into solid things like yogurt,
00:00:47:13 sour creams.
00:00:48:21 Sometimes they are very solid, like an aged Cheddar.
00:00:56:07 When you make a dairy product, there are many challenges.
00:01:01:12 One of the inconveniences of industrial operation
00:01:05:04 is that milk is not sterile when it comes to bacteria.
00:01:09:03 It's also not sterile when it comes to viruses.
00:01:12:22 Now, luckily for humans, most viruses in the environment
00:01:17:03 don't attack human species,
00:01:19:04 they actually attack bacterial species.
00:01:23:14 And viruses of bacteria are called bacteriophage.
00:01:29:18 If you used to be French like I used to be French
00:01:31:15 you have to say bacteriophage.
00:01:34:03 And if you're a dairy manufacturer
00:01:35:21 and you're using strep thermophilus
00:01:38:02 you buy industrial scale starter cultures,
00:01:41:22 It's a population of bacteria
00:01:44:06 that is put into a large vat
00:01:46:23 to start the fermentation process.
00:01:49:23 But occasionally those fermentation would fail,
00:01:52:19 because the bacteriophages,
00:01:54:15 those bacterial phages
00:01:56:05 would find it's way to the milk.
00:01:59:14 And when that phage is there,
00:02:00:21 it's gonna attack, eat and kill the bacteria
00:02:07:08 used to ferment the milk.
00:02:09:21 And then you get rotten milk.
00:02:12:17 Your case is a bad day.
00:02:14:15 That phage is a pain, it's very inconvenient.
00:02:17:06 (soft instrumental music)
00:02:23:04 So in the ongoing warfare between small viruses
00:02:26:09 that infect bacteria, this is a very dynamic arms race.
00:02:32:08 Viruses are very nimble, very small
00:02:35:13 they mutate very fast, they're in high numbers.
00:02:38:19 They don't need to do many things just need to infect host,
00:02:41:02 so that the simplicity of a virus is just unbelievable,
00:02:43:14 fantastic.
00:02:45:23 The host has to do all the work,
00:02:47:09 that's why viruses are so hard to fight.
00:02:56:12 I was a new employee working on a project,
00:03:00:00 that was industrially sponsored by company called Danisco
00:03:03:02 that was a very large food ingredient
00:03:05:07 manufacturing company out of Denmark.
00:03:07:14 So we wanted to understand what is in the genome
00:03:10:13 of those bacteria that make great yogurt and great cheese.
00:03:14:15 And how is it that they work.
00:03:16:21 I was like freshly minted PhD guy, right
00:03:21:05 that had expertise in doing Molecular Biology
00:03:24:04 in lactic acid bacteria.
00:03:26:04 One of my first jobs was to assemble those genomes.
00:03:29:04 Can we get the genome data?
00:03:31:00 And can we put the puzzle together?
00:03:33:07 And that answer is yes, about 98, 99% of it.
00:03:37:09 But when you come to the CRISPR, it breaks down.
00:03:43:07 A CRISPR is actually an array.
00:03:47:00 It's a piece of DNA that contains
00:03:50:00 two different kinds of parts.
00:03:53:22 One is the CRISPR repeat.
00:03:56:12 And the repeat is the defining core element of a CRISPR.
00:04:01:20 All those repeats are physically and genetically separated
00:04:07:11 by something called a spacer.
00:04:09:18 They are spaced out.
00:04:11:18 So essentially what a CRISPR array is,
00:04:14:18 a sequential combination of the CRISPR repeat, a spacer,
00:04:20:14 the CRISPR repeat, the spacer,
00:04:22:11 the CRISPR repeat, a spacer and so on and so forth.
00:04:26:07 The repeats are always the same.
00:04:28:14 So actually they provide very little information
00:04:30:17 with regards to where they come from
00:04:32:04 and what they do, because they're all the same.
00:04:34:21 In contrast the spacer sequences are all pretty peculiar.
00:04:39:04 They are different.
00:04:40:13 They are distinct.
00:04:42:14 By definition CRISPR is an acronym,
00:04:46:05 which stands for
00:04:47:01 Clustered Regularly Interspaced Short Palindromic Repeats,
00:04:51:18 It's kind of a mouthful.
00:04:52:23 But it's a very good descriptor.
00:04:55:12 So clustered means co-located together at one location.
00:05:02:02 Regularly interspaced regards the periodicity
00:05:07:01 and order with which different elements of CRISPR,
00:05:11:04 are positioned relative to one another.
00:05:14:18 Short CRISPR repeats by nature
00:05:18:21 are between 29 and 36 nucleotides.
00:05:22:12 Palindromic refers to the actual sequence
00:05:27:06 the DNA sequence within a CRISPR repeat,
00:05:31:04 is partially palindromic because they are pieces
00:05:34:18 within that repeat that compliment one another.
00:05:39:03 And then R stands for repeat.
00:05:40:08 So they're clustered regularly interspaced,
00:05:43:10 short partially palindromic DNA repeats.
00:05:47:03 (soft piano music)
00:05:51:04 And you had these beautiful visually intriguing
00:05:55:03 and mysterious array of repeats back to back
00:05:59:18 to back, to back, to back, 32 times of this chromosome.
00:06:03:09 And they would look interesting,
00:06:04:22 but we didn't have any clue as to what it did
00:06:08:03 and what the functionalities may be.
00:06:09:19 So, we just looked at it, it looked cool
00:06:12:18 and then we looked away.
00:06:14:17 And that was it.
00:06:15:16 That was like a very short lived,
00:06:18:01 one date only type event.
00:06:21:23 Back in the days people focused primarily on the repeat part
00:06:26:04 'cause it was the conserved part.
00:06:28:11 And ironically, people used to think
00:06:30:23 that the spacer part was junk.
00:06:33:06 It was a random gibberish that was inconvenient.
00:06:35:19 (upbeat music)
00:06:38:16 But by random chance, we saw homology to viral sequences
00:06:45:06 and we saw them repeatedly over and over
00:06:49:07 and over and over again.
00:06:52:02 So the key area of focus for a company like Danisco
00:06:57:20 is to develop great cultures that are resistant
00:07:02:06 to bacteriophage.
00:07:03:22 And one of those eureka moments was that,
00:07:06:11 the CRISPR genotype correlated with the grouping determined
00:07:11:19 by the phage sensitivity pattern.
00:07:14:19 So now we had two pieces of the puzzle,
00:07:16:14 one piece coming from the genome sequencing,
00:07:19:07 telling us that the repeats are awesome.
00:07:22:04 The spacers are unique and variable
00:07:24:12 and they show homology to viruses.
00:07:27:20 That's clue number one.
00:07:29:12 And at the other end of the spectrum,
00:07:31:11 we know that those harbored variable sequences
00:07:33:09 are super good to tell
00:07:34:19 what strains share similar bacteriophage susceptibility,
00:07:40:02 and we pulled those two things together.
00:07:42:05 (soft instrumental music)
00:07:46:17 So we sequence more and more and more strain.
00:07:49:11 And what actually happen is,
00:07:50:11 we sequence strains back in time,
00:07:53:01 back all the way to the eighties.
00:07:54:19 And when we compare and contrast them and align them,
00:07:57:23 they look almost the exact thing.
00:08:01:11 Across the whole length of those 2 million,
00:08:05:00 A T Cs and Gs together, they look 99.999% identical.
00:08:10:20 And one of those differences is right at the CRISPR locus.
00:08:17:09 One looks longer,
00:08:19:14 there's one more repeat and one more spacer.
00:08:23:13 And the amazing thing, was that some of those strains
00:08:26:22 that had evolved over time,
00:08:29:03 from the eighties and nineties and so on and so forth,
00:08:31:17 we could tell that the CRISPRs had changed,
00:08:36:00 within the timescale of the lab at Danisco.
00:08:43:08 What happened?
00:08:44:20 What is it?
00:08:46:10 And you dig into it, you dig into it and you dig into it,
00:08:48:08 so we will like look at the sequence,
00:08:50:14 look for matches with a sequence.
00:08:52:05 And of course, when we sequence those strains
00:08:54:21 of strep thermophilus,
00:08:56:12 we also sequence those painful phages
00:09:00:02 that make our work difficult.
00:09:03:01 So we have the sequences of the virus,
00:09:06:00 and we can tell that the small difference coming in
00:09:09:20 is a piece of DNA that is a hundred percent match
00:09:14:00 to that of the virus that was used to infect the host.
00:09:18:18 Now, it suggests, it doesn't prove anything,
00:09:22:07 that there may be a correlation between the CRISPR content
00:09:26:08 and the ability of the strain to be resistant to the virus.
00:09:29:12 And that prompted us to do a series of three experiments
00:09:34:04 to try to investigate whether there is or not,
00:09:37:20 the link between CRISPR and phage resistance.
00:09:41:13 So the first thing that we do is we try to repeat
00:09:43:17 what was done in the eighties and nineties.
00:09:46:15 When we take a strain, that same wild type strain
00:09:49:07 that is gets slaughtered by that phage,
00:09:51:17 and we expose that strain to the same phage,
00:09:54:04 what happens?
00:09:58:04 And then when it's kind of happy and actively growing
00:10:01:17 and multiplying and having the time of its life,
00:10:04:09 so to speak from the bacterial standpoint,
00:10:06:08 then you expose it to the phage, you let it incubate.
00:10:10:07 What's gonna happen is most of the cells are gonna die.
00:10:14:12 But if you give it more time
00:10:17:03 the smaller number of bacteria
00:10:19:00 become immunized against the virus.
00:10:23:09 So what you do is you let that grow over time,
00:10:27:03 and then you plate that on a petri dish
00:10:31:21 with great food, great comfort, and let it grow.
00:10:36:08 And multiply and multiply and multiply
00:10:39:06 and multiply enough to make a mountain of bacteria.
00:10:43:08 And if you look into it with enough resolution,
00:10:46:19 you will see a spot of a colony of bacteria
00:10:50:22 that have risen to resist.
00:10:53:00 And then what we do is we take those bacteria,
00:10:55:20 this one variant, and those guys we sequence.
00:11:00:10 So what we do is we monitor the CRISPR sequence,
00:11:04:07 that 33 repeats 32 spacers,
00:11:07:18 and we'll look at the CRISPR content
00:11:10:00 of the ones that make it.
00:11:11:23 We see repeatedly that when the strain becomes resistant
00:11:16:19 it has acquired a new spacer, and that spacer sequence,
00:11:21:16 the small piece of DNA that gets added on
00:11:24:14 is a perfect match to the phage that was used to do it.
00:11:28:17 So by then, we now know that there's likely
00:11:31:02 a link between the CRISPR spacer content
00:11:33:13 and phage resistance.
00:11:34:09 That was the first experiment, so we're pretty psyched.
00:11:37:15 It looks pretty convincing, but it's circumstantial.
00:11:40:04 We haven't proven anything, it's a suggestion,
00:11:42:23 it's a clue that in the process of becoming resistant,
00:11:49:12 it captured like a genetic Snapchat.
00:11:52:13 It captured a piece of DNA from the phage
00:11:55:11 that was used to attack it and kill it.
00:11:59:23 And then what we do is we go back to the host
00:12:03:01 and we do the same thing with a different phage.
00:12:07:00 And we see the same thing,
00:12:09:01 when we expose the same wild type parents strain.
00:12:12:17 It steals a piece of DNA from the phage
00:12:14:17 and extends the CRISPR.
00:12:16:17 And in a kind of a cool twisted experiment,
00:12:19:08 what we do is we give it a cocktail.
00:12:22:01 It's called a phage cocktail,
00:12:23:10 you blend different kinds of phages together.
00:12:27:11 Now we wanna see what the host is gonna do.
00:12:31:08 And the host picks up a piece of DNA
00:12:34:23 from each one of those phages.
00:12:37:03 We show there's a change
00:12:38:18 between the CRISPR content and phage resistance,
00:12:41:11 but we haven't associated the two.
00:12:44:12 They correlate, but we don't know the causality of it.
00:12:49:13 so you wanna show convincingly,
00:12:51:14 that the CRISPR spacer content determines
00:12:55:17 not correlates with,
00:12:57:14 but determines sensitivity or resistance to a virus.
00:13:01:21 (instrumental music)
00:13:04:14 So we went back with a second series of experiments.
00:13:07:06 If we synthetically add a spacer
00:13:11:17 without the host ever seeing the virus,
00:13:14:21 what happens?
00:13:17:14 We have to clone
00:13:19:18 using recombinant DNA technologies and techniques,
00:13:23:02 a synthetic CRISPR repeats spacer unit.
00:13:27:15 And we transfer that DNA, that plasmid into the host.
00:13:35:04 So now if you add a spacer,
00:13:37:18 you gain resistance to the phage
00:13:39:17 without ever having seen the phage.
00:13:43:12 We've proven when you add the spacer synthetically,
00:13:47:09 you reprogram the CRISPRs,
00:13:49:13 so to speak, you become resistant.
00:13:57:08 The CRISPR locus, these repeat spacer,
00:13:59:09 repeat, spacer, repeat array
00:14:02:12 is oftentimes accompanied by adjacent sequences.
00:14:08:02 CRISPR associated sequences called Cas genes.
00:14:14:13 And those Cas genes likely code Cas proteins
00:14:18:14 that are physically and genetically next to the CRISPR
00:14:23:19 and CRISPR together with Cas
00:14:26:06 constitute the CRISPR-Cas system.
00:14:29:05 (upbeat music)
00:14:33:14 So the question is,
00:14:35:00 if we tinker with those Cas genes
00:14:37:17 and we disable them
00:14:39:12 does it have an impact on the function of CRISPR
00:14:44:01 in providing resistance against the virus?
00:14:47:01 So essentially you insert a piece of DNA
00:14:49:15 in the middle of a gene to disrupt it
00:14:52:00 to make it not function.
00:14:54:03 We didn't know what it was gonna do.
00:14:55:23 It was a true unknown.
00:14:57:02 It was a true discovery type experiment,
00:15:00:06 if we knock this gene out and inactivate this piece of DNA,
00:15:05:22 that we don't know what it does
00:15:07:14 in a system that we don't know how it works.
00:15:10:05 Is it gonna have any impact or not?
00:15:12:00 And then what impact is gonna have if it does?
00:15:14:22 What we observed, just amazing.
00:15:17:10 We show that when you inactivate Cas9 genetically,
00:15:22:01 you lose the ability to be resistant to the virus
00:15:26:01 though the CRISPR spacer is there.
00:15:28:23 The spacer just being there is not enough.
00:15:32:10 This implicates, Cas9 in resistance to the virus.
00:15:38:20 That was like, I'm so excited.
00:15:41:09 I can't believe it like that's cool, that's cool.
00:15:43:08 Like, I can talk about it all day.
00:15:44:20 Like, it's really cool.
00:15:45:16 Like I remember looking at the plate
00:15:47:11 coming in that morning,
00:15:49:05 opening the incubator and looking at it like,
00:15:52:04 man, it's gone.
00:15:53:06 Like, it's gone, like by tinkering with this one element,
00:15:56:19 we've broken the CRISPR.
00:15:59:16 And we had no idea that it was gonna do anything.
00:16:03:15 We now know Cas genes and the Cas proteins they encode
00:16:08:17 are necessary for the CRISPR locus to work.
00:16:13:19 And then in September, 2006,
00:16:17:09 I can go to my management at Danisco and tell them,
00:16:20:07 okay, guys, we have now proven
00:16:24:10 through the experiments that we did
00:16:26:05 that CRISPR provides adaptive immunity in bacteria.
00:16:30:12 And eventually we published our paper in March, 2007.
00:16:34:18 Exactly 20 years after the original observation
00:16:40:19 of a CRISPR locus in E. coli in 1987.
00:16:45:20 It took 20 years for CRISPR to make it from first observed
00:16:51:14 to substantiate it from a biological standpoint.
00:16:55:19 The 2007 paper from the theoretical standpoint
00:16:58:18 is so impactful because what this is,
00:17:01:04 is establishing CRISPR
00:17:02:20 as the adaptive immune system in bacteria,
00:17:05:17 which nobody thought they have.
00:17:07:16 It's this molecular equivalent in some ways
00:17:10:15 of the antibody antigen reaction
00:17:12:20 that we have as complex vertebrates.
00:17:16:06 It's this trans generational implication,
00:17:21:01 that there is a evolutionary system and mechanism
00:17:24:19 that adapts to its environment
00:17:26:19 and adapts to the phage and strategically evolve over time
00:17:31:18 to overcome a threat.
00:17:33:17 (instrumental music)
00:17:37:11 So, as a food scientist
00:17:40:03 and as a foodie, in many ways,
00:17:41:19 I like to think of science
00:17:45:22 as an example of how food works.
00:17:51:03 And also food as an example of how science works.
00:17:57:06 And in many ways to get the masterpiece like CRISPR,
00:18:03:02 it takes a lot of key ingredients.
00:18:05:18 You have to have a great recipe in and of itself.
00:18:09:07 You have to have a great cook, a great chef.
00:18:11:04 You have to have the kitchen, the utensils,
00:18:15:00 the raw ingredients.
00:18:15:23 You want some good wine.
00:18:19:09 You need a bit of salt, but not too much.
00:18:22:02 And then you need to actually do it,
00:18:23:05 just having those ingredients themselves,
00:18:25:04 doesn't guarantee masterpiece whatsoever.
00:18:28:08 And in the end, there's things you can't control,
00:18:31:05 like the ambiance.
00:18:33:10 And for a masterpiece to be enjoyed,
00:18:36:21 you need to be in the right company
00:18:39:02 and have the right setting and have the right context.
00:18:42:19 And that's what's so compelling about CRISPR
00:18:44:09 is there's so many fantastic pieces to that story.
00:18:49:16 The history of CRISPR,
00:18:52:11 the people involved, the scientists involved,
00:18:54:12 the companies involved.
00:18:56:12 The applications.
00:18:59:03 And that's what makes for a compelling story
00:19:02:09 and a compelling journey.
00:19:04:08 (soft instrumental music)

This Talk

Speaker: Rodolphe Barrangou

Audience:

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

Recorded: July 2017

Talk Overview

The discovery that CRISPR functions as an immune system to protect bacteria against viral infection was made at Danisco, a company looking to improve yogurt production. Rodolphe Barrangou tells the story of his discovery, which laid the groundwork for the gene editing revolution.

Speaker Bio

Rodolphe Barrangou

Rodolphe Barrangou studies beneficial microbes, focusing on the occurrence and diversity of lactic acid bacteria in fermented foods and as probiotics. Using functional genomics, he has focused on uncovering the genetic basis for health-promoting traits, including the ability to uptake and catabolize non-digestible carbohydrates. He spent 9 years at Danisco-DuPont, characterizing probiotics and starter cultures,… Continue Reading

Credits

Elliot Kirschner (Executive Producer, Science Communication Lab): Executive Producer
Ron Vale (Executive Director, Janelia Research Campus): Executive Producer
Sarah Goodwin (Executive Director, Science Communication Lab): Executive Producer
Steven Tyler: Editor
Gb Kim (Illustrator, Explorer’s Guide to Biology): Illustrator
Derek Reich: Cinematographer

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