Prof Martin Jínek - CRISPR Cas Genome Editors - from Bacteria to Biotech (25.02.2021)

hello everyone welcome to another tcss talk this talk will be about an hour long and afterwards there will be a q a session both will be live streamed on youtube and also record it if you have any questions either write them into the youtube chat or you can ask them personally during the q a session the link to the organization will be posted into the youtube chat just before it so if you want to join the zoom call then new to your youtube live stream and join with your full name now please let me introduce our today speaker professor martin nienek professor marty nine is a researcher at the department of biochemistry university of zurich he completed his undergraduate studies in nature sciences at trinity college university of cambridge and he got his phd at the european molecular biology laboratory in heidelberg robertson conducted his postdoctoral research at the university of california in berkeley with professor jennifer dodna they were studying crispr systems and their use as a gene editing tools forest research 2020 nobel prize in chemistry was awarded to professor dona and professor charpentier in today's talk professor younique will be speaking about crispr cast genome editors please welcome professor unique professor enig the stage is yours okay thank you very much america and andrea for the invitation to give a lecture within the framework of the trinity college science society i'm really happy to be able to do that so yeah hello and good evening from from switzerland i will share my screen to um hopefully it's it's up now and it's working uh so yes um thank you very much once again for the for the invitation um it was really a no-brainer to to agree to give this talk as i have a connection to trinity and so today what i will try to do is to give you an overview of what crispr cass genome editing is and how we basically came to where we are now uh with the technology how basically a discovery of the technology um was well how the development of the technology was was driven by discoveries initially made in bacteria and then in in test tubes and then led to the uh to the technological revolution that these new genome editing tools uh give us so before i get on with that however i um would again like to thank you for the invitation because um you know i have a connection to trinity i was an undergraduate student at trinity college between 98 and 2002. um i must confess that you know there were no digital cameras and no smartphones in those days so there are actually very few photos of me from from that time and the photo that you see on the screen is actually from a few years later when i when i came back to trinity to pick up my free uh m.a degree this was in back in 2004 or 2005 and i had a digital camera like that already okay so anyway so um yes it's it's an absolute pleasure to be um to be back at trinity virtually uh uh at least and uh to to uh uh share with you my uh my journey through uh through crispr and through uh through genome meditating um as was already mentioned i'm now based at the university of zurich and so what i would like to do here is basically acknowledge my research group it's a it's a wonderful group of phd students and postdocs and and also undergraduates who come and do their bachelor and master projects in our lab and if there's one thing that i'm proud of in my research is it's it's really this the fact that you know i i have the privilege and the the good fortune to work with really fantastic people so my research is basically firmly rooted in my long-standing passion for rna for rna as a molecule and for its various molecular activities in cells and in particular i'm interested in the molecular mechanisms of protein rna complexes and various molecular machines that work on rna and this in particular in the context of genome defense mechanisms and their applications as genome editing technologies and so where all of this basically comes together to [Music] to a large extent is uh is crispr both as a as a genome editing technology but also as a as a bacterial immune system that is used to find viruses in bacteria and so um as as you're probably aware uh we're really in the midst of a genuine editing revolution um where the emergence of the crispr cast genuine editing technology is something that allows us to modify the genetic information in cells and organisms in a way that's simple fast and you know precise and also quite quite cheap relatively speaking is something that gives us unprecedented powers for not just basic research but also for biotechnology and from molecular medicine and in particular the the application of crispr cast genome editing for gene therapy is something that i think myself and a lot of people are of course very excited about another aspect of genome editing and or crispr cass and this is something that i'll hopefully come to in the in the last few minutes of my talk is that you know this technology is really giving us for the first time the power to modify our and our own dna in a way that can be really transmitted to the next generation and with this power of course i would say comes the responsibility to use this to use this technology wisely and so um since 2012 when when crispr cast genome editing emerged this has really become a technology or a method for the masses so to speak the due to the reduced costs and the simplicity of the method it has now basically thoroughly revolutionized not just basic research but it's uh making a lot of breakthroughs in in biotechnology as well as as as well as in molecular medicine and really giving us um the ability to do things um in the genomes of cells and organisms that until a few years ago were not really possible and so the the the basic principle of of crispr cast genome editors is that at the core of the technology we have protein based enzymes that act as dna dna nucleases as dna cutting enzymes but whose specificity in terms of the dna sequence that they recognize that they they can cut is determined by the sequence of a guide rna that can be made and then associated with the with the cast nuclease to to program it to to target a specific sequence in the genome of the cell and so what basically these genome editors allow us to do is to cut dna with quite a high degree of precision in cells or in organisms and generate something known as a double strand dna break and this is basically a type of dna damage that the cells encounter all the time and have for that purpose involved dna repair mechanisms and it is basically this combination of the dna cutting and then the subsequent dna repair that can be exploited for the uh for the for the installation of genetic modifications in dna because the dna repair mechanisms can be basically exploited and manipulated to some degree to achieve specific dna repair outcomes that result in modifications so we have the combination of dna cutting with endogenous dna repair which then leads to the modification that we would like to achieve and depending on which cells we do this in and depending on which dna repair pathway can be exploited um we can basically do on one hand rather simple things like you know knocking out genes or making mutations at specific positions in in the dna or do more complicated genetic modifications such as inserting dna sequences into a specific locus in the genome or things like you know correct various mutations at a specific site in the genome and so again very very simply put the the crispr cast genuine editing technology really allows us to you know modify dna as if we were basically modifying a piece of text on a computer and so uh the the discovery of the of the mechanism underlying these these genome editor nucleases and and their rna-based programmability is something that as was already mentioned was recognized with the award of the of the 2020 nobel prize in chemistry to emmanuel sharpened here and to jennifer dowd now and um you know i was i was very uh i was very privileged i guess and uh fortunate to be basically part of the team that was behind the work that led to the nobel prize so in 2012 we were basically the team that that characterized the the first rna guided dna cutting enzyme and basically set off the uh the genome editing revolution um on its on its trajectory so what i would like to do today and uh in in this lecture is to basically give you an overview of crispr cast genome editing about its mechanisms about its applications and so i'll begin by telling you a little bit about where crispr cass came from i will then follow up by explaining a little bit about how these crispr genome editors how these dna cutting enzymes work at the molecular level i will talk a little bit about how crispr cast genome editing can be implemented in cells and organisms and then finally how it can be used to treat uh human diseases and so i'll begin by telling you where crispr crispr actually came from and so as i already alluded in in at the beginning of my talk uh crispr has its origin in uh prokaryotic cells in bacteria and archaea where they uh where the system basically provides a kind of a genome defense mechanism if you will or an adaptive immune system that provides resistance against viruses against mobile genetic elements such as plasmids and others and the first sign or the first indication first i guess discovery of the crispr system was was made back in the 1970s and 1980s when the first genomic sequences of bacterial genomes became available and where it became clear that in bacterial genomes we basically had these specialized repetitive sequences uh consisting on one hand of basically repeats that had a specific sequence in the specific length which were interspaced with uh non-repetitive sequences that became known as spacers and so what basically defines the crispr system in a bacterium or in an archaea is basically this presence of this repeat spacer uh pattern in the genome and this is basically then where crispr actually thought it's got its name because it's based on this uh it's this it's an acronym based on clustered regularly interspaced short paranormalic repeats which basically describes the uh the kind of the appearance of these of this loci in in the genome and so for about 20 years the function of these crispr loci was really a mystery until in 2007 a group of researchers at uh at a food company called danisco made the discovery that these crispr systems actually functioned as a acquired resistance mechanism against viruses so as a kind of a primitive adaptive immune system if you will and so what became basically clear was that these spacers which i'm showing here as these colored squares in the in the bacterial chromosome basically had sequences that were matching the dna sequences found in viruses and that the presence of a spacer in the genome of a bacterium was basically sufficient to make that bacterium resistant against the virus that contained the same sequence in its dna what also became clear around that time was that the crispr system was adaptive meaning that bacteria would be able to take dna from a previously unknown virus incorporated into its chromosome to its in into its own genome and acquire resistance against that virus and so this mechanism basically um can be essentially thought of as a kind of a vaccination card for the uh for the bacterium bacterium basically makes it makes makes notes or makes a makes a permanent record of its previous encounter with a virus in its own dna and then uses it to recognize the virus and to target it and eliminate it the next time the bacterium is basically infected by that virus or comes into contact with it with its dna now at the molecular level this is where basically things got very interesting for me because it became clear that there's rna involved in the mechanism so what happens is that this space of repeat structure in the genome is transcribed as a long rna precursor and then processed into individual small rna molecules each of which basically then contains a sequence that's derived from one of the spacers and what these rnas then do is that they associate with proteins that are produced by the transcription and translation of the so-called crispr associated genes which are always found together with this with this array in a in a crispr system and they form basically targeting complexes in which the the crispr rna functions as a molecular guide basically the rna uses the information encoded by the sequence of the of the spacer to basically target the the cast protein to the dna of the virus using base pairing interactions and so in this way um the the rna basically provides the targeting capability and marks the dna of the of the virus for destruction typically by some kind of a dna nuclease degradation mechanism and so again you know if we want to kind of summarize the function of the of the crispr system it's it's basically that first the uh bacterium will take the dna of the virus and insert it into its own genome to you know effectively memorize the encounter with the virus and then based on that information it will generate gyn rnas that will program these cast proteins for the recognition of the viral dna by base pairing interactions and this basically recognition mechanism is then coupled to dna targeting by nucleases to basically destroy the dna by by cutting effectively and so my contribution basically to this uh in in the early days of christopher genome editing was that we um and by we i mean myself christophe kelinski and jennifer downer and emmanuel schampeter and our colleagues we discovered that in a subset of these bacterial crispr systems we really had a protein known as cas9 that combined basically all of these functions in that it acted as a dna cutting enzyme that was programmable by by these guide rna molecules and that at least in its natural form basically associated with two rna molecules one of them being the crispr rna that provides the targeting information and then the other being an rna molecule known as tracer rna which basically acts as a kind of a co-activator it's basically required for the crispr rna to be recognized and to associate with uh with the cas9 protein and to program it and so it's the combination basically of the crispr rna and the tracer rna and the cas9 protein then enables the bacterium to target the viral dna that's complementary to the targeting sequence in the in the guide rna now we realized in the in the course of our biochemical studies that we could basically take uh the [Music] dual guide rna structure and we could simplify it by fusing the crispr rna and the tracer army together to generate basically a single molecule guide rna structure and so basically reduce the complexity of the system even further to the point where we have basically just one protein to do the dna cutting and one guide rna format with which we can we can program this protein to target the sequence that we want to modify and so the idea of this one guide rna one protein system is then the basis of the genome editing technology as we as we know it nowadays and so what i'll talk about now is basically how these genome editor nucleases actually function at the molecular level and this is something that i personally am very interested in and it's something that my own research group in zurich has has focused on over the last eight years where we were basically trying to understand the molecular mechanism of these of these nucleases uh using a combination of chemical and structural approaches and so we started uh working on on cast nine um in this respect because this is at least mechanistically this is this is a fascinating enzyme um so as already mentioned it basically has this dual rna guide structure uh that it uses to find a matching sequence in the target dna and this is basically uh dependent on the base pairing interactions between a 20 nucleotide uh sequence in the guide rna so 20 letters of the rna have to match 20 letters in one of the strands of the dna and if this is the case then the enzyme will basically catalyze the cleavage of the dna at this position within the target site and it will do it by basically independently catalyzing the hydrolysis of the two dna strands and for that it has two two nuclease domains so two active sites in this enzyme to catalyze the two dna strand cleavage events now this is actually a little bit more complicated because um the process of dna targeting and cutting also requires that the dna contains an additional sequence motif outside of the of the target site and this is referred to as a something called protospacer adjacent motif or pam and for the most widely used cas9 genome editor this is basically a sequence of this consensus so ngg where n can be any of the four dna bases and so this has to be present basically in the dna otherwise the system will not recognize the the target and will not be able to cleave it even if it's perfectly complementary to the to the guide rna model so this is this is an additional requirement basically for the for the dna cutting and of course to be able to recognize in the the dna in the first place you have to have the the match between the guide rna and the target dna particularly uh in this you know pan-proximal side of the of the of the duplex but this in itself is not again not enough it's it's sufficient for dna binding but if you need to cleave the dna then you also need to have base pairing in the in this part of the rna dna duplex at the other end and again there's a there's a there's an explanation for why this is uh in the molecular mechanism of the enzyme and so we were very interested in in these aspects of the targeting mechanism and we wanted to gain insights into this and in my lab we do this by basically taking a structural approach we use structural methods like x-ray crystallography and nowadays also increasingly cryo-electron microscopy to uh basically determine structures of these enzymes in various states and then to basically um you know learn more about uh about the molecular mechanisms that we can then test using using other approaches either by chemical or assays or by physical assays and and so on and so um our work on cas9 basically led to one of the first structures of the protein in complex with the guide rna and the target dna and what i'm showing here is basically a a 3d printed model of the molecule in white we have the cas9 protein in red we have the guide rna and then in the two shades of blue we have the two strands of the dna target and so what you see here in the middle is basically the rna dna duplex that the enzyme makes and that's positioned right in the middle of the molecule kind of surrounded by uh by various parts of the the cas9 protein one other interesting thing that you uh maybe can can see here is that when cas9 binds to dna it actually bends it by almost 90 degrees it's also something that we think contributes to this process of separating these two strands of the dna from each other and forming the rna dna duplex and so the beauty of having molecular structures and by the way this structure was determined by my very first phd student caroline anders is that you can really zoom in and get get all the details and so in our case one of the details and one of the highlights of the structure was basically the mechanism by which this enzyme recognizes this panel this critical element of the of the recognition mechanism where we could basically show that this was done by specific amino acid residues in the protein contacting the dna bases using using hydrogen bonding interactions and so we've studying cast 9 but we also in addition started working a few years later on cast 12a which by that point emerged as a as an alternative genome editor nuclease again it's a it's an rna guided dna cutting enzyme that is found in a different class of crispr cast systems and it has somewhat complementary and again very interesting properties so unlike cast 9 it has a slightly different pan specificity the pam is located at the other at the other end of the target site with respect to the to the polarity of the guide rna it is a naturally occurring single rna guided system so there's no tracer rna in the type five crispr cast systems where cast 12a is found it cleaves the dna by a slightly different mechanism because it only has one nuclease active site and so again there are very interesting aspects of the mechanism that we then continued studying using structural and biochemical approaches and so one thing that i wanted to share with you is again in this case a movie that's based on a couple of x-ray crystal structures that um we solved in my laboratory uh this was the work of a postdoctoral fellow called dan schwartz and based on which we were able to again make it kind of a somewhat idealized movie of um of well depicting how um how the the dna targeting mechanism basically works and this is hopefully something that you can see so we have basically the cas9 protein and the guide rna and now the enzyme is basically depicted uh as basically sliding along a double-stranded dna to search for for its target and the search basically begins with looking for the for the pam sequence and once the pam sequence has been located by the enzyme this basically triggers the unwinding of the two dna strands and the formation of the rna dna duplex which basically forms in this kind of a zip like manner from one end to the other and when this occurs then um the dna is basically then positioned within the protein in a way that the protein can catalyze the cleavage of the two dna strands in a in an ordered sequential mechanism and so by basically elucidating the the structures of these enzymes we have been able to you know highlight some of the mechanistic differences as well as commonalities that uh these uh that these two molecular systems have and um our goal in in this in this whole enterprise is to basically be able to use these insights not only to improve our understanding of these systems in the context of the biology of crispr's cat systems in prokaryotic cells but also as a way to contribute to the continued development of crispr cast genome editing technologies we hope to be able to use structure guided engineering of the of the proteins as well as the guide rnas to address some of the constraints that these that these systems have uh things like uh improving the specificity of these systems and minimizing their of target activities expanding the targeting space by basically modifying the pam specificities of these enzymes which you know constrain these these enzymes to only specific sequences in the genome uh and and to do other things like you know improve the efficacy of these of these enzymes and specific cell types and and others and so how do we actually go on about implementing crispr cast genome editing and so again i will come back to the to the slide that i showed you at the beginning where i told you that what we basically do in genome editing is to exploit the rna programmability of these dna nucleases to make double strand breaks and then relying on the endogenous dna repair mechanisms to uh then introduce modifications into the dna and again very in a sort of somewhat simplified way we have two main dna repair mechanisms one called non-homologous and joining which can be used to make either short insertions or deletions in the dna because this is a kind of a quick and dirty mechanism that doesn't doesn't really care about whether the double strand break is is repaired with any degree of precision or not and so this can be basically used to make indels in the dna or to knock out genes by basically disrupting the open reading frame of the protein coding gene on the other hand we have homology directed repair which is a set of dna repair mechanisms that rely on having a homology template for the repair process and so by providing that that repair template exogenously together with the cas9 protein and the guide rna we can basically trick the dna repair mechanism to use that dna dna template and to basically modify the dna if that template is designed in a way to you know insert a piece of dna into this into the genome or to overwrite part of the genetic sequence with a with uh with something else and so by combining basically um cas9 or cas12a based dna cutting with homology directed repair we can we can really do quite complicated and very precise genetic modifications and so this idea of using dna cutting enzymes to do genome editing is actually something that was already known before crispr came along and there were already quite powerful technologies to do that but the main drawback was that they were based on designed artificially engineered proteins in which a dna nuclease and non-specific dna nuclease was fused to sequence specific dna binding proteins or to the combinations of sequence specific dna binding protein domains and so the main drawback of these two technologies zinc finger nucleases and talents was that you know every time you wanted to target a different sequence in the genome you had to design a new protein and even though the design of these proteins was quite modular this was still a tricky thing to do and so when crispr cast 9 and later some of the other genome editors came along this process was greatly simplified because all you had to do now was to simply change the sequence of the guide rna to reprogram the enzyme to target a different sequence in the genome and so very shortly after our discovery in 2012 a number of studies were published showing that this could indeed be done in eukaryotic cells and we were also involved or i was also involved in one of these studies showing that indeed cas9 could be used together with these guide rnas in eukaryotic cells for genome editing so in our study what we did was we basically took pieces of dna so dna plasmids encoding on one hand the cas9 protein and on the other the guide rna and introduce them into cultured human cells in a petri dish and a few days later we basically isolated the genomic dna from uh these cells and uh started sequencing individual uh clones uh from um from these uh from from this dna and we were basically starting to see these telltale signs of dna repair happening exactly at the site that we were targeting with the enzyme so we were basically recovering clones where part of the dna sequence was deleted as well as we had one clone where there was a there was an insertion um at this site and so this was basically for us the first indication that we were getting dna repair uh following dna cutting by the rna-guided cas9 protein in cells and so this then basically led to the implementation of the of the technology so that basically nowadays all you need to do really to target a specific gene in the in the genome is to essentially modify the 20 nucleotides at the five prime end of the guide rna just basically write in the sequence that you want the genome editor to to recognize in the genome and and deliver the protein and the kind rna potentially also together with a with homology repair template into cells and for this you nowadays have actually various options the simplest way as i already told you was to simply deliver both components in the form of dna that's encoding them and then relying on the cells to basically express uh the protein and the guide rna to form the uh the complex inside for certain organisms or certain experimental systems you can also deliver these components in the form of rna but something that's becoming more and more common in the field is basically to simply uh pre-assemble the protein rna complex in in a test tube using a recombinant cas9 protein that you can buy from a company and a synthetic rna that you can either make yourself in a laboratory or just buy again from from a company that's specialized in oligonucleotide synthesis and then delivering these uh pre-assembled complexes into cells using using various methods if the cells are big enough you can do this by micro injection but typically for cultured cells you would nowadays do it by electroporation basically delivering an electric pulse of the cells to temporarily disrupt the membrane and make them make them take up the um the protein rna complex and so this direct delivery of the protein rna complex is something that is is quite nice because um first of all you're not delivering any dna into cells so both the cas9 protein and the guide rna basically have a limited life span in in those cells and that means that you also end up with fewer of target effects because there's basically um less time during which the cas9 protein and the guide rna are active and during which they can not only modify the the target site that you want to actually modify but also potentially or accidentally modify the you know other sites in the genome that are partially complementary to the guide rna so the the rnp delivery not only is useful for reducing this of target activity but also can improve the the efficiency of the process to begin with because the protein rna complex is active from the moment it gets into cells so nowadays um you know if you want to basically modify the genome of a cultured cell line um what you need to do is basically get your recombinant cas9 protein design a guide rna based on the sequence that you want to you want to target so you would look for the presence of a pam you would basically then take the sequence of the dna next to the pam plug that sequence into the guide rna have that guide rna made and then deliver it into cells together with the cas9 protein as well as with any kind of a dna repair template that you want to use for a specific modification and then treat the cells using this this electroporation approach so put cells into this cuvette and then simply zap it with with electricity again very simply put and if you do this and you design the the repair template that in a way that you know not only introduces a piece of sequence into the into the genome but also introduces a restriction endonuclease site you can then very very simply you know check for the presence of that piece of dna and for its insertion in the dna by isolating the genomic dna of the cells and just doing a simple restriction digest and so um as again as i already mentioned um you know when we think about implementing crispr cast genome editing um there are still some constraints that we would ideally like to be able to address and to improve the basic capabilities of the technology and in particular at least in you know in the context of my work we would really like to provide structural insights that can be used to to do this and so one of the concerns surrounding the use of cas9 particularly in the context of genetic therapies is the specificity already quite quite early on it became clear that these uh that these genome editor enzymes are perhaps not as specific not 100 perfect and that they basically can target other sequences that are similar but not perfectly matched to the guide rna for cast nine you know sequences containing up to five mismatches can be tolerated by the enzyme to some extent and so there's quite a lot of effort now in the field aimed at improving the specificity of the enzyme and to basically reduces its off-target activity and this is something that has led to the development of you know engineered high fidelity variants of the basic naturally occurring enzyme where by targeting specific amino acid residues in the protein that would normally contact the dna um you can basically make the system much more sensitive to mismatches between the rna and between the guide rna and the dna um by basically perturbing the let's say the the thermodynamics and the kinetics of the the dna cutting reaction and so by now there are basically several of these high fidelity enzymes that are that are available and that have reduced off target activity another development in the field is basically the emergence of crispr based molecular tools that enable you to do genome editing without having to cut the dna in the first place and this is um this is a technology that was developed in david lew's lab primarily at harvard where the technology basically relies on the fusion of cas9 protein with a an enzyme that can basically catalyze the chemical conversion of one base into into another so in this case the the cas9 protein is not used to cut the dna but is used in its inactive form just simply as an rna guided dna binding platform that will deliver this base modifying enzyme to the dna so what this cytodine deaminase does it will basically take a c and turn it into a u such that and when the dna is replicated that you will be decoded and you know and then a will be inserted in the opposite strand during dna replication so we can basically use this to turn one base pair into another in the dna without the need for dna cutting another recently developed technology again attempts to do editing without cutting by fusing again a nuclease deficient form of the cas9 protein with a reverse transcriptase and using a modified guide rna as a as a template for the reverse transcription reaction where the uh the dna um once it's been nicked by the uh by the cast iron protein will be um will be extended uh by the reverse transcriptase activity and then then lead to you know insertion of sequences into the site based on the on the sequence of the modified guide rna so this approach has been uh it's been termed prime editing and again it's a it's a again another development in um in this molecular toolkit for genome editing and so finally uh what i would like to talk about a little bit is uh about the potential of crispr cast for genetic therapies and ultimately the idea here is that we could use crispr cass genome editing tools to really correct disease-causing mutations in the human genome and to do this um you know very precisely and without having to rely on you know kind of the previous uh approaches in gene therapy which relied on gene augmentation basically on this idea that you know we would um essentially rescue the function of a defective gene by inserting another copy of the gene somewhere in the genome so instead of this what crispr cast would potentially allow us to do is to simply go back to the to the defective genome exactly at the site uh sorry go back to the defective gene exactly at the site of the in the genome where it's where it's found and just simply correct the disease causing mutation an example a classic example of a situation like this is you know the sickle cell disease where a single nucleotide mutation in the beta-globin gene leads to the production of a defective hemoglobin variant that you know causes the disease phenotype and so one could basically approach this by by simply correcting that that mutation in um in that gene and so uh for this again one can think about two uh two approaches to do this one would involve delivering the crispr cast nine or crisper cast 12a or any of these technologies directly into cells or organs or tissues in a living human patient so basically doing in vivo genome editing and this is something that is being developed and where the technologies basically will use things like viral vectors or lipid nanoparticles to basically deliver these components into into cells another approach which uh is proving to be a bit more simple and at least in the short term is ex vivo genome editing where the goal is to basically do genome editing in cells that have been extracted from the patient modified in a laboratory and then reintroduced into the patient once the genetic defect has been has been corrected in these cells and here in the future what is going to be potentially very powerful is the combination of genome editing and the ipsc technology so induced pluripotent stem cells where you know we could conceivably you know take fibroblasts or other other cells from the patient reprogram them to make ipscs in in the laboratory then modify those ipscs differentiate them into the specific cell type that you would like to like to like to get and then introduce those cells back into the patient so there's there's a lot of of course effort now focused on implementing these two approaches for specific genetic diseases so i will give you a few examples of this so there are by now you know dozens of clinical trials using crispr cast genome editing this is an example very recently of a genome editing strategy to treat beta beta globin deficiencies such as sickle cell disease or beta thalassemia by basically disrupting the beta-globin gene which carries the mutation and and basically reactivating um the expression of gamma globin so the fetal globin subunit in in adult erythroclasts and so so this is something that is already in clinical trials and it will be of course very very interesting to to see how this how this will develop and i think the you know the initial results are incredibly incredibly promising another area where ex-vivo genome genome editing in cells is making a big impact is in the process of engineering immune cells for treating various forms of cancer so in this case the goal is not to correct you know a genetic defect but rather to reprogram the immune cells of a patient to basically make them recognize cancer cells specifically so this is basically by inserting specific receptors on on the surface of these these t cells and also uh by you know introducing other genetic modifications that make these t-cells um um you know irresponsive to various forms of inhibition and so on and so reprogramming t cells to target cancer cells is going to be again another powerful technology and powerful application of crispr cass if we talk about in vivo editing here again there's quite a quite a lot going on i think overall the field is maybe not as modest advanced as for ex vivo genome editing on the whole but there have been a number of really breakthrough studies where by delivering crispr cass components into into model organisms which had which serve as you know models of human genetic diseases you know other researchers were able to really get some spectacular results uh an example of this was uh basically these three studies showing that uh in a mouse model of duchenne muscular dystrophy you know really debilitating uh neuromuscular genetic disease um you know the treatment basically with uh viral virally delivered cast 9 was able to basically restore muscle function in these in these animals and so perhaps unsurprisingly you know there's there's a lot of excitement about the prospect of using genome editing for therapeutics there are a number of companies that are active uh in in this area and you know a number of different diseases that are being actively actively investigated and so it's going to be something that will be you know coming to the hospital near you very soon and this is also reflected i guess in the enthusiasm of the of the stock market for these uh for these companies but you know before we can get there um i think we will be have we will have to address some of the some of the some of the constraints of the of the of the technology and if it's of its yeah specific applications so uh you know the ongoing work in in the field is really focused on things like improving the specificity on improving the delivery for many of the diseases and many of the indications the delivery will actually be the the more tricky uh part of the of the therapeutic strategy than the actual editing uh but also for the editing one one of the key areas is to really get a better way of controlling the dna repair process or to avoid the endogenous dna repair process altogether by using some of these technologies that don't necessarily rely on them in the first place and so this is something that you know is going to revolutionize genetic therapies and molecular medicine in the future and so finally uh something that i want to mention at the very end is the ethical side of it because until now we've talked about um you know editing uh human patients and basically doing the editing in somatic cells but um of course there's uh there's the possibility of using crispr cast genuine editing also for editing in the human germline and this is something that's you know a very ethically controversial aspect of crispr cass genome editing this of course became the topic quite early on in 2015 when the first genome editing experiments were done in human embryos and then again was reinforced by the reports in 20 2018 of the first genome edited babies being made in china and so of course this is something that is creating a lot of controversy in the field and it's it's what i said what i see as a as a good development is that there is basically an ongoing debate about what would be the responsible path forward for the use of genome editing there have been you know global international summits and and ongoing discussions it's clear that for now uh you know research and and the use of crispr cast genome editing for somatic gene therapies is something that should should go ahead um the use of germline genome editing is something that should not be done for two main reasons one that there is uh currently well the technology is not mature enough yet to ensure that it can be safely and efficiently used but mainly because so far there hasn't really been a a widespread societal consensus on on the use of this technology for general and genomic in many parts of the world this is something that's prohibited by law uh and uh you know not not possible and so it's clear that um we will have to have ongoing discussions and and it's something that you know the scientists cannot really decide for themselves this is something that really involves the whole society and and all the different stale stakeholders in this so with this i i would like to conclude my my talk uh i hope that i've given you an illustrative overview of the journey that crispr cass has taken from bacteria all the way to biotech and to micromedicine and i would like to basically finish on on this note by by saying that you know the the discoveries that led to the development of genome entity this was really basic research this was not something that um initially aimed to do to do genuine medicine this came out of you know people being curious about bacteria about how they defended themselves from from viruses and how these you know how these uh sophisticated molecular systems actually function and so if if there's basically a lesson in this it's it's this that if you think you you have a great idea for a technology nature has probably been there before you and so it's it's worth basically investing into basic science to um you know to look for interesting and potentially useful molecules in nature to then repurpose for other for other applications in technology so with that thank you thank you very much for listening i'll be happy to answer any questions and uh yeah once again thank you to the organizers for inviting me to give the talk uh at trinity college it's great to be back after after all these years so thank you you know professor enac many thanks for the great talk and now it's time for a q a session so drag can you please post the link yeah the link for to join our zoom collis in the youtube chat so please mute the youtube live streaming beforehand and join with your full name if you have any questions and meanwhile there are some quests okay um hello can i fire off or sure yes hi yeah thanks very much for your talk martin i was i was um wondering about the mechanism of cast 12 binding to dna because he showed that for cas9 there are some arginine fingers binding to the bomb motif yes so is it is it conserved is it the same in cast 12 um this mechanism are perhaps unsurprisingly different given that the pams are also different in in cast 12 the mechanism is some is is actually based more on the readout of the shape of the dna rather than on the actual recognition of specific bases in the panel so for cast 9 the pam is t rich and you know if you have a stretch of or a series of t residues and or 80 base pairs then the minor group of the dna is basically a bit more narrow yeah that's something that the the enzyme can can recognize because then you know it has a shape that's that's basically complementary to the to the shape of the dna and and this is basically why um cast 12 enzymes in general actually prefer to try these um in other cas9 proteins that have different prime sequences there you again see different different amino acid residues contacting the pumps and different combinations this is something that's also been exploited to to actually engineer new variants of cast iron that recognize other pamphlets so you are looking at the different evolutionary solutions in the lab as well like pursuing the catalytic mechanism and trying to improve the current like state of the art so so we already um so we already looked at the the fan recognition mechanism earlier so back in 2015 um you know the first engineered variants with different pam specificities were developed um and then very shortly after that we um we determined structures of those and structural information kind of fed the development of yet another generation of genome editing variants of time not in my laboratory but in other laboratories um and and so our current uh our current uh focus or current interest is actually the uh um the off-target activities i'm actually very interested in you know what it means when when you have a dna that's not perfectly complementary and how how how come you know how come this enzyme actually is quite tolerant of mismatches in certain parts of the rna dna duplex what kinds of mismatches and so on so this is something that is an ongoing project well on that note i was i was going to ask about your opinion on the prime editing technique because i i i was trying it in the lab during the summer and i got like almost zero efficiency in editing with this and it also looks a bit dodgy because there are so many improbable events that need to occur before you actually get the edits do you think this this is usable in in like everyday lab work or stuff like that so i'm you know i don't have a i don't have a direct experience with it with this uh with this method um you know i'm you know i'm i'm more of a basic basic researcher than a yeah energy developer but um you know the feedback or the kind of the uh from my interactions with some of my colleagues who have uh who have used this um you know it's it seems to work quite well in certain cases and sometimes it doesn't but and and right now it's it's still too early to tell why exactly so i see yeah um i've i've heard you know people having quite good experience maybe it's a bit you know a bit dependent on on the specific specific targets and you know specific cells and so on okay thanks thanks very much and then perhaps final question um i'm i'm i'm trying i find it difficult to conceive of how crisp binds in the context of chromatin because if you think about it in bacteria it's obvious that there is no there are no nucleosomes and stuff like that so it can bind directly to viral um dna invading the cell whereas in mammalian systems for instance you've got the repressive effects and the environment of chromatin so how does it overcome it or if you know the kinetics of how it actually scans the dna i haven't like perused the literature on that so um you know this is some something that has not really been uh completely resolved yet at least in in vitro it's it's clear that nucleosomes actually are quite refractory to you know cast line binding to dna and and cutting it and actually if you want to cut dna that's that's within chromatin at least in in vitro you have to make the enzyme you know target essentially the linker dna between individual nucleosomes yeah i think what what happens in cells is that um you know the chromatin is actually not static it's it's it's it's quite dynamic it's remodeled um of course in some parts of the genome you know more than than in others um but um at some point you know the the dna site that you want to target basically becomes exposed and essentially nucleus and free if you if you will um you know especially if you think about you know proliferating cells where you have to replicate dna anyway and you know chromatin is remodeled in the course of dna replication and so on so so it's it's something that um you know is probably probably a factor here because at least in vitro if you have if you have nucleosomes on the dna and they're kind of you know by under the specific place then it's actually quite hard for cas9 to access the dna displacement so do you think if you fuse gasoline to some nucleosome remodeling activity like uh enzyme could you improve the efficiency well this is something that people have been trying to do yeah okay yeah also as a way of controlling uh transcription energy basically to transcriptionally activate or repress a locus by you know delivering um you know either chromatin remodelers or nucleosome modifying enzymes yeah basically transferases all right so now we have a few more questions in the chat so first one is how do you make the cell do homologous recombination to repair the dna but you wanted to repair it that way or how do you make the cell do nhea to repair the dna when you want to do gene knockouts right so so this is something that you don't necessarily have um a lot of choice about at least you know without anything you know without the help from from something else in in i get you know in most cells the non-homologous unjoining is basically the predominant dna repair pathway the homology directed repair is you know at least you know in my understanding is something that's really only active in in proliferating cells where where dna is is being replicated because you know the purpose of you know you can only really repair by homology if you if you have the template to begin with and you know you have to template basically when when you've replicated your dna so so homology directed repair is of course most active in proliferating cells and is is less active or completely absent from uh post-mitotic you know differentiated cells so this is why it's for example very difficult to do um homology-based editing in things like neurons or you know other differentiated cell types and so you can improve the efficiency of of homology directed repair using several tricks one is for example to use chemical inhibitors or [Music] you know other ways of inhibiting or repressing non-homologous and joining another way is to uh for example um synchronize the cells and basically deliver the the casino and guide rna at a specific point in the cell cycle there are other ways you know we had a we had a collaboration with the lab of gerald shrank here in zurich where you know we attempted to improve the process by basically covalently linking the cas9 protein and the homology repair template and this also has an effect so so there are there are ways to to bias this towards um towards um you know one repair pathway or the other but um but still this is something that's you know a bit of an unresolved unresolved problem with the technology or you know one of the limitations okay then there was a succinct question from the same person also what specific function does the pma sequence actually have interaction with the cas9 i understand that pma is required to cleave dna but why well it's basically required because when the enzyme binds the dna it it looks for the pan first and the palm really establishes the if you will the register of the dna sequence [Music] against the against the guide rna basically when when cast 9 or cast 12 have located the pam then um we we still don't know what happens in the really in the very early stages of this process but but the assumption is that this initial interaction with pam either distorts the dna or does something to the dna so as to facilitate the initial separation of the two strands so that once the strands have been separated the target strand can start base pairing with the with the guide rna because i mean the problem is that or you know the beauty of these enzymes is that they they essentially unwind the dna without any energy input you know they just use the thermal energy in the system and then rely on the fact that uh you know overall the process is kind of thermodynamically downhill that for every baseband in the dna that you break you make a base pair with the dna and so if you do this you know one base pair at a time you know the the overall say activation energy barrier is is lowered but still you have to get to the first opening of the dna and this is basically coupled to the process of of pan recognition so so the pam is basically necessary to open up the open up the dna and then to form the structure that allows the the guide rna to to base between the dna then another question what is the main principle behind the off-site targeting is it the non-perfect homology between the guide rna and the target sequence or the enzyme and itself so so this is to a large extent basically driven by by the tolerance for for mismatches between the rna and the dna and specific positions and um also the type of the mismatch seems to seems to have a an influence on how how bad you know how badly of target activity is then and around i think it's maybe related to does dna modification take modification or accessory proteins affect the targeting of the gene um um well it's it's a bit of a yes yes and no question so at least the you know the the dna modifications that we encounter in eukaryotic dna like you know um cytosine methylation and so on this doesn't really affect um the the basic recognition mechanism there are however certain dna modifications that viruses can make to their own dna that may that can make the dna refractory so there are certain bacteriophages they they glucosylate they glucosylate dna bases and depending on the position of the glucosylation you can you can inhibit cast iron activity uh as it turns out castor oil a is not so it's not so sensitive to this and and and there is we think sort of a structural explanation for it because you know it all comes down to how much how much how much room there is in the in the major group of the dna and whether that you know somehow sterically clashes with the with the protein and so on but for the for the eukaryotic modifications this is this is not a major factor as far as i'm aware and last question from the chat could you comment something about non-biomedical applications of crispr cars so this is something that i obviously haven't really really had the time to go into but but that's um again you know another another big area of interest where um you know one could think of using using cas9 in you know synthetic biology to you know generate new new microbes that are genetically engineered microbes that would have interesting properties you know be able to you know do biosynthesis of new molecules and so on so that's something that you know genetic engineering i mean in in in many organisms the genetic engineering is is um already possible now but but but there are um there are organisms where this could make a make a a big difference and it is being used to you know engineered biosynthetic pathways and so on so that's one one area and the other is of course you know modifying potentially plants or animals for for use in in agriculture but again there's quite a lot of quite a lot of interest quite a lot of development um but again you know this is also something where there's a there's quite a lot of controversy um particularly in europe and you know in the context of you know wood crisper you know would organisms modified by crispr for example fall under um you know the current gmo regulations um and and so on where i think there's there's a bit of a difference between europe and the us and the rest of the world in how this is being approached great so lukas do you have many any more questions or um yeah can i have one more question please i so you showed us the crystal structure of gas 9 and i mean if i look at it it seems like there could be many heterogeneities introduced by the differences in the dna positioning and rna positioning so i was wondering how you made it crystallize in the first place or if if there were any issues with that or if you how did you stop it at the intermediate which you're like showing in the crystal structure um yeah so so to make to make cas9 crystallized first of all um you know we were um trying to prevent the dna from being cut so we were using a catalytically inactive mutant but you can also crystallize dna with the catalytically active protein uh the issue is that you know if you want to crystallize something you have to i mean the molecules will crystallize only if they if they form contacts you know that lead to the formation of a crystalline lattice and this is where um you know this is a process that's not so easy to control you can you can you know you can try to facilitate it by especially if you work with nucleic acids by you know engineering the the dna and the rna in a way that for example you know make sticky ends where the bases could you know somehow come together and this would lead to the formation of crystal contacts um somehow um i think you know in for the cast line structures we were actually quite lucky that um you know the the specific sort of dna design that we took just accidentally led to the formation of a crystal contact between one loop in the guide rna and basically the part of the um you know one side of the of the duplex where you know there's a basically a blunt end and you have base bases base pairs kind of stacking on top of each other for the cast 12 structures um there we had to use a trick and really modify the dna to have overhangs and and this led to the formation of basically a kind of a continuous stack of molecules along one of the directions of the one of the axes of the of the crystalline matters so so this is something that you know um you kind of have to do in in in x-ray crystallography but nowadays um you know with the advent of uh cryo-electron microscopy this is something that we don't really have to worry about anymore and so this is why uh cryo-em has been so powerful also in in the crispr field because these molecules are they're big enough to be imaged in the electron microscope so this is where everybody's kind of moving including us okay thank you very much looks like there are no more questions so professor inik many thanks for your great talk again and for answering the questions once again also for the invitation it's uh it's been a great pleasure and uh yeah hope to have a chance to see you at some point when yeah we'll be looking forward to it i'm conditioned to permit i haven't been to cambridge in a few years it would be great to come back all right take care you too bye bye bye bye and next thursday there will be a talk by professor harry anderson from the university of oxford and professor anderson will be talking about molecular nanostructures and their unusual properties the talk is going to start at the usual time at quarter past six of the uk time so thank you for attending the talk and have a nice evening

Channel: Trinity College Science Society

Views: 1,278

Rating: 4.9166665 out of 5

Keywords:

Id: znlX1p3Tubs

Length: 83min 0sec (4980 seconds)

Published: Tue Mar 02 2021