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Jennifer Doudna (UC Berkeley %2F HHMI)- Genome Engineering with CRISPR-Cas9 - Contenido educativo
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Hi, my name is Jennifer Doudna from UC Berkeley, and I'm here today to tell you about how we
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uncovered a new genome engineering technology.
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This story starts with a bacterial immune system.
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That means understanding how bacteria fight off a viral infection.
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It turns out that a lot of bacteria have in their chromosome, which is what you're looking
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we're looking at here, a sequence of repeats, shown in these black diamonds, that are interspaced
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with sequences that are derived from viruses.
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And these had been noticed by microbiologists who were sequencing bacterial genomes, but
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nobody knew what the function of these sequences might be until it was noticed that they tend
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also occur with a series of genes that often encode proteins that have homology to enzymes
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that do interesting things like DNA repair.
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So it was a hypothesis that this system, which came to be called CRISPR, which is an acronym
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for this type of repetitive locus, that these CRISPR systems could actually be an acquired
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immune system in bacteria that might allow sequences to be integrated from viruses, and
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then somehow used later to protect the cell from an infection with that same virus.
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So, this was an interesting hypothesis, and we got involved in studying this in the mid-2000s,
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right after the publication of three papers that pointed out the incorporation of viral
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sequences into these genomic loci.
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And so what emerged over the next several years was that, in fact, these CRISPR systems
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really are acquired immune systems in bacteria.
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So, until this point, no one knew that bacteria could actually have a way to adapt to viruses
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that get into the cell.
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But this is a way that they do it.
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And it involves detecting foreign DNA that gets injected, like shown in this example,
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from a virus that gets into the cell,
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the CRISPR system allows
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integration of short pieces
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of those viral DNA molecules
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into the CRISPR locus.
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And then in the second step
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that's shown here as CRISPR RNA biogenesis,
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these CRISPR sequences
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are actually transcribed in the cell
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into pieces of RNA
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that are subsequently used together with proteins encoded by the Cas genes,
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these CRISPR-associated genes, to form interfering or interference complexes
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that can use the information in the form of these RNA molecules
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to base pair with matching sequences in viral DNA.
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So, a very nifty way that bacteria have come up with to take their invaders
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and turn the sequence information against them.
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So, in my own laboratory, we have been very interested for a long time
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in understanding how RNA molecules are used to help cells to figure out how to regulate
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the expression of proteins from the genome.
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And so this seemed like also a very interesting example of this.
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And so we started studying the basic molecular mechanisms by which this pathway operates.
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And in 2011, I went to a scientific conference and I met a colleague of mine, Emmanuelle Charpentier,
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who is shown in this picture on the far left.
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And Emmanuelle's lab works on microbiology problems, and they're particularly interested
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in bacteria that are human pathogens.
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She was studying an organism called Streptococcus pyogenes, which is a bacterium that can cause
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very severe infections in humans.
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And what was curious in this bug was that it has a CRISPR system, and in that organism
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there was a single gene encoding a protein known as Cas9 that had been shown genetically
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to be required for function of the CRISPR system in Streptococcus pyogenes.
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But nobody knew at the time what the function of that protein was.
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And so we got together and recruited people from our respective research labs to start
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testing the function of Cas9, and so the key people in the project are shown here in the
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photograph.
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In the center is Martin Yinek, who was a postdoctoral associate in my own lab, and next to him in
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the blue shirt is Krzysztof Czajlinski, who was a student in Emanuel's lab.
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And so these two guys, together with Inez Fanfara, who's on the far right, a postdoc
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with Immanuel began doing experiments across the Atlantic and sharing their data.
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And what they figured out was that Cas9 is actually a fascinating protein that has the
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ability to interact with DNA and generate a double-stranded break in DNA at sequences
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that match the sequence in a guide RNA, and in this slide what you're seeing is the guide
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the guide RNA and the sequence of the guide in orange that base pairs with one strand
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of the double helical DNA, and, very importantly, this RNA interacts with a second RNA molecule
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called tracer that forms a structure that recruits the Cas9 protein.
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So, those two RNAs and the single protein in nature are what are required for this protein
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recognize what would normally be viral DNAs in the cell, and the protein is able to cut
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these up, literally, by breaking up the double helical DNA.
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And so, when we figured this out, we thought, wouldn't it be amazing if we could actually
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generate a simpler system than nature has done by linking together these two RNA molecules
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to generate a system that would be a single protein and a single guiding RNA.
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And so the idea was to basically take these two RNAs that you see on the far side of the slide
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and then basically link them together to create what we call a single guide RNA.
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And so Martin Jinek in the lab made that construct and we did an experiment,
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a very simple experiment, to test whether we truly had a programmable DNA cleaving enzyme.
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And the idea was to generate short, single guide RNAs that recognize different sites
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in a DNA molecule, this circular DNA molecule that you see here.
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And the guide RNAs were designed to recognize the sequences shown by the red bars in the
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slide.
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And the experiment was then to take that plasmid, that circular DNA molecule, and incubate it
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with two different restriction or cutting enzymes.
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one called SAL1, which cuts the DNA sort of upstream at the far end of the DNA in this picture,
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in the gray box, and the second site being directed by the RNA-guided Cas9 at these different sites,
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shown in red.
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And a very simple experiment, we did this incubation reaction with plasmid DNA,
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and this is the result.
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This is... what you're looking at is an agarose gel that allows us to separate the cleaved
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molecules of DNA, and what you can see is that in each of these reaction lanes we get
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a different sized DNA molecule released from this doubly digested plasmid that... in which
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the size of the DNA corresponds to cleavage at the different sites directed by these guide
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RNA sequences indicated in red.
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So this was a really exciting moment, actually.
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So, it was a very simple experiment that was kind of an a-ha moment when we said we really
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have a programmable DNA-cutting enzyme and we can program it with a short piece of RNA
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to cleave essentially any double-stranded DNA sequence.
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So, the reason we were so excited about an enzyme that could be programmed to generate
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double-stranded DNA breaks at any sequence is because there was a long-standing set of
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in the scientific community that showed that cells have ways of repairing double-stranded
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DNA breaks that lead to changes in the genomic information in the DNA.
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And these... so this is a slide that just shows that after a double-stranded break is
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generated by any kind of enzyme that might do this, including the Cas9 system, those
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So, these two-stranded breaks in a cell are detected and repaired by two types of pathways,
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one on the left-hand side that involves non-homologous end-joining, in which the ends of the DNA are
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chemically ligated back together, usually with the introduction of a small insertion
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or deletion at the site of the break, and then on the right-hand side is another way
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a homology-directed repair in which a donor DNA molecule that has sequences that match
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those flanking the site of the double-stranded break can be integrated into the genome at
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the site of the break to introduce new genetic information to the genome.
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And so this had given many scientists the idea that if there were a tool or a technology
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that allowed scientists or researchers
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to introduce double-stranded breaks
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at targeted sites in the DNA of a cell,
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then together with all of the genome sequencing data
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that are now available,
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where we know the whole genetic sequence in a cell,
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and if you knew where a mutation occurred
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that causes a disease, for example,
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you could actually use a technology like this
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to introduce DNA that would fix a mutation
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or generate a mutation that you might like to study in a research setting.
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So, the power of this technology is really the idea that we can now generate these types
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of double-stranded breaks at sites that we choose, as scientists, by programming Cas9,
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and then allow the cell to make repairs that introduce genomic changes at the sites of
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these breaks.
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But, the challenge was how to generate the breaks in the first place.
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And so a number of different strategies had been produced for doing this in different labs.
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Most of them, and I'm going to show two specific examples here,
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one called zinc finger nucleases and the other TAL effector domains,
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these are both programmable ways to generate double-stranded breaks in DNA
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that rely on protein-based recognition of DNA sequences.
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So, these are proteins that are modular and can be generated in different combinations
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of modules to recognize different DNA sequences, requiring... so, it works as a technology,
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but it requires a lot of protein engineering to do so.
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And what's really exciting about this CRISPR Cas9 enzyme is that it's an RNA-programmed
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protein.
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So, a single protein can be used for any site of DNA where we would like to generate a break
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by simply changing the sequence of the guide RNA associated with Cas9.
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So, instead of relying on protein-based recognition of DNA, we're relying on RNA-based recognition
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of DNA, as shown at the bottom.
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And so what this means is that it's just a system that is simple enough to use that anybody
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with basic molecular biology training
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can take advantage of this system
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to do genome engineering.
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And so, this is a tool, then,
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that really, I think, fills out
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an essential, previously missing component
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of what we could call biology's IT toolbox,
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that includes not only the ability to sequence DNA
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and look at its structure,
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we know about the double helix since the 1950s,
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and then, in the last few decades,
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it's also possible to use enzymes like restriction enzymes
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and the polymerase chain reaction
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to isolate and amplify particular segments of DNA.
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And now, with Cas9,
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we have a technology that enables facile genome engineering
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that is, you know, available to labs around the world
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for experiments that they might want to do.
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And so this is a summary of this...
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of the technology.
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It's a two-component system.
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RNA-DNA base pairing for recognition,
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and very importantly,
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because of the way that this system works,
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it's actually quite straightforward
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to do something called multiplexing,
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which means we can program Cas9
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with multiple different guide RNAs
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in the same cell
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to generate multiple breaks
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and do things like cut out
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large segments of a chromosome
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and simply delete them
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in one experiment.
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And so this has led to a real explosion
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in the field of biology and genetics,
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with many labs around the world
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adopting this technology
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for all sorts of very interesting
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and creative kinds of applications.
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And this is a slide that's actually
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almost out of date now,
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but just to give you a sense
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of the way that the field
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has really taken off.
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So, we published our original work
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on Cas9 in 2012,
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and up until that point
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there was very little research
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going on on CRISPR biology anywhere.
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It's a very small field, and then you can see that starting in 2013 and extending until
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now, there's just been this incredible explosion in publications from labs that are using this
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as a genome engineering technology.
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So it's been really very exciting for me as a basic scientist to see what started as a
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fundamental research project turn into a technology that turns out to be very enabling for all
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sorts of exciting experiments.
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And I just wanted to close by sharing with you a few things that are going on using this
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technology.
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So, of course, on the left-hand side, lots of basic biology that can be done now with
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the engineering of model organisms and different kinds of cell lines that are cultured in the
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laboratory to study the behavior of cells, but also in biotechnology, being able to do...
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to make targeted changes in plants and various kinds of fungi that could be very useful for
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different sorts of industrial applications, and then of course in biomedicine with lots
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of interest in the potential to use this technology as a tool for, you know, really coming up
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with novel therapies for human disease I think is something that's very exciting and is really
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something that's on the horizon already.
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And then this slide just really indicates where I think we're going to see this going
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in the future with a lot of interesting and creative kinds of directions that are coming
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along in different labs, both in academic research laboratories, but also increasingly
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in commercial labs that are going to enable the use of this technology for all sorts of
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applications that we, many of which we couldn't have even imagined even two years ago.
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So very exciting.
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And I want to just acknowledge a great team of people that have been involved in working
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on the project with me, and we've had the, you know, terrific financial support from
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various groups as well, and it's been a pleasure to share this with you.
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Thank you.
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- Subido por:
- Francisco J. M.
- Licencia:
- Reconocimiento - No comercial - Compartir igual
- Visualizaciones:
- 63
- Fecha:
- 26 de diciembre de 2021 - 13:39
- Visibilidad:
- URL
- Centro:
- IES ALPAJÉS
- Duración:
- 16′ 42″
- Relación de aspecto:
- 1.78:1
- Resolución:
- 1280x720 píxeles
- Tamaño:
- 109.35 MBytes
