Jennifer Doudna (UC Berkeley / HHMI): Genome Engineering with CRISPR-Cas9

Jennifer Doudna (UC Berkeley / HHMI): Genome Engineering with CRISPR-Cas9


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

100 thoughts on “Jennifer Doudna (UC Berkeley / HHMI): Genome Engineering with CRISPR-Cas9”

  1. ALL playing with genes is very dangerous!!!
    Genes are developed in nature for millions of years, and humans, thinking they know everything, now are making and spreading CHAOS!!!

  2. The first real cut and paste tool for our "software". Let's hope "genome software developers" don't create nastier bugs when trying to solve "old bugs". Debugging tools for this type of coding will create many moral problems. Responsible "developers" are hard to find. Hopefully the complexity of the "whole system" will prevent "small inconvenient bugs" from creating drastic consequences.

  3. Thanks Jennifer for sharing. This has helped me immensely with my studies in Biotechnology . Excellent presentation!

  4. I have to perform CRISPR technology in my project and I have found that this video has explained CRISPR beautifully!
    Thank you for your time in creating and uploading this.

  5. The hard part seems to be how to do this in live cells if correction of genetic diseases is the ultimate goal. Doing this in a lab setting whereby DNA can be altered in vitro is one thing. Controlling CRISPR/Cas9 cleavages and sequence repairing in vivo sounds like 20-30 years away.
    Anyone know who owns this technology? There's an IPO coming out this week called Editas Medicine. I wonder if this group is part of that startup.

  6. Dr., I have a question. Can Crispr take a person with a bad gene and fix all the DNA in the body at once? So if someone has the gene for breast cancer, can you take a woman in her early 20s and fix this bad gene that causes breast cancer? If not now, could it be done in the future? I ask because I don't understand if Crispr only can fix problems before birth on the FETUS or if it works on adults as well. The fact I have to ask this is a good example of a huge disconnect when scientists try to explain things to the public. You guys should interview regular people first, talk about things like Crispr, and then do a video like this. Thank you.

  7. hi!can u plz suggest me any amazing recent research in field of genetics and biochemistry ……like crispr cas

  8. DNA Signal Processing: Towards a hardware-in-the-loop CRISPR viral defense metaheuristic evolution

  9. Her evil Highness Doudna FRANKENSTEIN – Hybrid mother and "Creatress" of future gene manipulated inhuman generation. Welcome to the club…

  10. I might sound ignorant because I don't know enough about this pathway… but if it is possible for a entire chromosome to be spliced out, why haven't genetic diseases like down syndrome been cured yet? If someone could tell me the implications preventing such feats that would be great. This area of study is truly quite interesting.

  11. also her last name reads as Do U DNA ? a fascinating breakthrough but sounds like the beginning of every horror sci-fi movie ever made…

  12. I realy can't understand how the gRNA binds to the cas9, I mean when have the CAS9 and the gRNA that we have just synthesized,which are both two separate macromolecules,what should we do to bind the two together properly .

  13. للاسف انا ما بعرف الغة انجلش وياريت واحد يترجملي لان الفديو باين عليه ممتع للغاية

  14. Does anybody know if i can study in the field of human gene therapy if i choose Biotechnology as a major this year? if not, do you have a better idea? i live already in germany

  15. These "scientists" think they're so clever messing in what God made and claiming it be their own. It's completely based upon narcissism and ego. The sheer conceit of these f*****g morons almost exceeds their sense of entitlement..
    When they unravel the physical codex of life and start messing with it. Guess what's going to happen in reality? They're going to lose the ability to return to that base and God created code. Why? Because they can't possibly unravel every aspect of it. It's designed to self destruct when interfered with. Not the first or 2nd generation – but with those afterwards. Perhaps a couple of decades or so.
    They will then end up in an ever diminishing circle of trying to do what? Fix what they have permanently broken. And all of it because of money and avarice.
    They should be put on trial for messing with what keeps us alive. Physically.

  16. I believe that when a cell is engineered to produce a "foreign" protein, the end product might be different from the intended peptide or protein, if the enzyme to cleave the signal peptide is not present in the endoplasmic reticulum of that cell. What a powerful tool, have been found, let's hope that it will be used for healing people! Congrats to all involved in this major breakthrough!

  17. Thank you so much @ibiomagzine. just one question what is PAM sequence prior to cleavge site? Is it necessary for specific cleavge?

  18. We really are a species of editors. The expressions "People never change." and " "It's a choice to stay the same." will need to be re-evaluated and qualified. When the implications of this technology become applicable, we had all better be much more philosophically capable of justifying EVERYTHING we do and say because genetics and fate will not longer carry the weight of loosely defining our elusive existence. All things will be programmable, we just need a sustainable value system on which to base our programs.The faithful will have heavy hearts of guilt, the faithless will need to program even their ethics to match their editorial decisions. It's fascinating to see humanity become a race of editors, from our photographic documentation of our lives in video and picture form to the very fabric of our genetic being, we are editors. It's no wonder fantasy and media content have had such an impact on the development of our personalities and ethics. To edit the world according to our imagined desires is paramount for us, whether it be making life like the movies or programming genes.

  19. Very interesting, very promising for future generations, hopefully not used for changing the man created by God, Adam and Eve took the tree of good and evil. now the new generations are trying to take the tree of life but God did not allowed. He does not like a evil and eternal creature, He is preparing a new creation with goodness inside them, obedient and saint to ensure an universe without conflicts nor sadness, He promised and he will accomplish, maybe this uncovering​ is part of that plan. I'm not again science.🙏

  20. Crispr godness ! but the efficacy of this technology is not very high. The Ngago/gDNA technology maybe a new effective way to edit gene.

  21. It feels as though they want to lead the research. Kudos on discovering. But they hide layers and speak in ways that others could not move forward with their work without them having foremost access to the knowledge first. Open source is they key to true evolution…..

  22. If I had a choice I'm OK with aging and dying, it's a natural process for me because it's giving my body back to nature but I'm hoping this really helps the future generations like my great great grandchildren to live very long lives, free of disease. I hope it becomes a success!

  23. I just got introduced to CRISPR-Cas 9 system, and I am genuinely amazed at how simple yet intricate this system is. Hats off to you and your team for finding this breakthrough. I think you have firmly carved your name into the history books of biological research.

  24. Thanks for very nice and well demonstrated video lecture on CRSIPR Cas-9 mechanism and its novel applications.
    Once a Bacterium is infected by a Bacteriophage, what type of enzymes cleave the viral genome within bacterium, to be remembered by Bacterial genome for recognition in future by CRISPR-Cas9 complex. I mean a detailed mechanism of Adaptive immune system adapted by Bacteria.

  25. This is not good at all… this information will NEVER be used for good and will ultimately end humanity in favor of some engineered being who will get wiped out by their robot overlords anyway. We don't need to improve our skills at living the good life!!! We already are and there are already 7.6 BILLION of human parasites wasting the planet away!

  26. Dos any body notice that she is discribing the process but not showing the scale of her find do u people notice the size of a DNA and she do not even explain how much of a pill well effect a person it like a person taking cocain how that little bit can effect and elephant

  27. And she dos not even explain what you can take to help a person out like a plant base example when it's winter u eat soup there is something in soup that would help u in winter so what can a person take to help him or her self in life

  28. Didn't You People already know by now? Capitalist Theory, abhors Problem Solving. When perpetual Problem "Maintenance" is much more Financially rewarding. CRISPR, is a so called Disruptive Technology, to the Established Order. Perhaps, even the most So, EVER!…..I just love a good Revolution!

  29. At 7:40 would be better to have used an acrylamide gel to resolve short DNA fragments, rather than an agarose gel. Bands would be razor sharp not fuzzy.

  30. Marvelous presentation of a fascinating topic. I would vote for you to get the Nobel prize considering the massive importance of your discovery. Glad to see my old alma mater is nurturing such famous people. I studied there in Physiology and took Organic Chemistry with Melvin Calvin. I'm a retired doctor and doing animations of CRISP Cas9 in Cinema 4D. What an exciting field you are in and what great work! Thank you for what you have done.

  31. If the CRISPR bacterial system is incorporating selective bits of foreign DNA, does this mean this mechanism won't work on RNA viruses? Without a RT enzyme in the bacterial cytoplasm how would it incorporate RNA genomic sequence?
    ?

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