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Studying And Fighting Pathogenic Bacteria with the Help of Crispr

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Studying And Fighting Pathogenic Bacteria with the Help of Crispr
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Cas systems have emerged has a powerful biotechnological tool. The Cas9 protein is a RNA-guided nuclease that can be easily reprogrammed to target any sequence of interest. Our work focuses on the development of CRISPR-Cas9 tools to edit bacterial genomes and control gene expression. In particular, we investigate how these tools can be used in high-throughput screens to perform functional screens. Recently we have also shown how CRISPR system can be used as sequence-specific antimicrobial. The Cas9 protein can kill bacteria when directed to cut in their chromosome. Guide RNAs can be programmed to kill Bacteria carrying antibiotic resistance or virulence genes specifically, and the CRISPR system can delivered to bacterial populations using phage capsids.
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so thank you very much for the opportunity to present my work in this confirmed that I've really enjoyed so far so I'm going to show you what we've been doing with system CRISPR in in in bacteria I know we're using them both to develop tools to to study and who to fight pathogenic bacterium so I first want to give you a bit of history about this CRISPR systems so they basically were first discovered but not understood in 1987 by this Japanese group was just sequencing this I up gene and just don't stream of the genes I've found this interesting sequence where you had this repeats that were interspaced by this sequence that looked sort of random and the size of the repeats and of these variable regions was a very very well conserved at the time of course the didn't understand at all what the sequence is where and you should look at the citation report for for that that paper basically it was published in 87 but it's got almost no citation citations until 2007 so and 715 hasn't been updated but it's probably up there now so what what happens in in 2007 is basically this paper so that's a landmark paper in the field of CRISPR it's basically the first experimental evidence that this CRISPR loss I actually provide acquired immunity against bacteriophages and the CI what they did in that in that paper is working this strain of streptococcus thermophilus the interesting thing also to note is that this this paper didn't come out from an academic group but rather from the industry and from a company called Dennis Co that was later bought by dipole that was developing ferments for lactic fermentation for yogurt production cheese production and these people as other speakers before I've noted a big problem with viral contamination that can ruin the batch production so they always try to select strains that are resistant to bacteria phages and when doing so basically they noticed that the CRISPR locus was changing and that it was capturing new sequences coming from the phages that were using the challenge and so when a CRISPR capture the specific spacer so froth at the sequence coming from a phage it becomes resistant to the fade that matches this this sequence so that was a very interesting discovery
so since 2007 as a field has moved at quite incredible pace and we now understand a lot about this CRISPR systems and this is basically the picture of other work so first I should also say that CRISPR itself stands for clustered regularly interspaced short palindromic repeats so that's very complex acronym and that was current actually by bioinformaticians so but that explains very well the structure of this of this slow side where you have these clusters of repeats that regularly interspaced and repeat themselves frequently contain short palindromes so hence this long CRISPR name and that is that when you have a bacteriophage injecting its DNA in the cell at some frequency the CRISPR system thanks to some proteins known as cast for CRISPR associated is able to capture a piece of DNA from the phage and integrate it along with the new repeats in the CRISPR locus once it's captured this this information in this adaptation phase is then able to use this information to fight infection by phages similar phases and the way it does that in this immunity phase is by transcribing the CRISPR locus in the first precursor on is that then processed into some small crisper in a and those small crispr RNA are actually complex with cast proteins that are going to be able to cut and degrade sequences of malagos to the crispr RNA so basically the CRISPR is building of memory of past infection and then using that memory to fight infection by similar sages I want to go just a bit more in details about one specific CRISPR system that's a type 2 C CRISPR system from streptococcus pyogenes and there's a good reason for that is this system is the one that is know mostly used for biotechnological application of of CRISPR and you see that this whisper system contains actually forecast gene cast main cast one casts to see sn2 does the CRISPR array was actually six spacer and repeats Island and the interesting part here is that it also has another small RNA involved in this process that's called a tracer RNA that stands for trans activating crispr RNA and this three cast genes here case one gets to see sn2 actually involved in the capture of new sequences for the CRISPR system and the cast 9:13 itself is about carry out so all immunity step from these systems so the role of this tracer RNA is actually also very important in this system what it does is that it's complimentary to the sequence of the repeat so when you transcribe the primary transcript here from the CRISPR array the tracer arena can then advertise making duplex RNA with each of the repeats and that duplex RNAs and recognized by cast nine and by the host RNAs three and that processes the primary transcript into the smaller crispr RNA and after this process you basically end up with this complex as cast nine and these two RNAs crispr RNA and the trace RNA and all the elements of this complex are through the essential for the function of the system so once you have generated these complexes nucleoprotein complex what happens is that it's going to basically look for possible targets we talked to the surveillance complex that's constantly scanning the DNA in the cell looking for Amala G's with the crispr RNA but first what the complex is actually scanning for it's looking for small motif in this case the motif is mg g and thus known as the Pam motif for proto space or adjacent motif and when it once cast nine finds this GG motif then it starts to unwind the double helix and pair it with a crisper Rene if there is some match then it can completely pair the full length of the of the crisper remains that triggers a conformational shift in the protein that brings two catalytic domains in contact with the double Alex introducing a double strand break in the in the target DNA and that can then lead of course to the degradation of the target DNA so the way the system works has basically been very well described by as a group of a manual Chaplaincy and Jennifer Doudna and that got them to get this breakthrough price last year and mingle with some only wood stars like a Cameron Diaz and the CEO of Twitter here so it's good to know that you should do exciting science you can meet your movie stars so what have we
been doing with with this system it'll you said so we're a microbiology group and we work on both understanding so where's this CRISPR system working bacteria but also developing technologies and today I'm going to mostly talk about the technological aspects and the first technology we developed using this is a strategy to edit the genome of bacteria and the strategy here that we employed so I'm showing here what we did for the for in e.coli we introduced a plasmid carrying cast 9 in the cell and then we can electroporated together another plasmid that is programmed with a CRISPR that will target here a gene we want to modify at the same time we introduced a specific mutation we want by translator operating a single-stranded illegal that carries the mutation one to introduce and we don't do that just in any strain we do that here in this heme sixties restraint from the lab of Donald Court that expresses a lambda red recombination machinery that allows to basically recommend this only go to high frequency and then the CRISPR system can basically select this mutation by killing all the cells that did not introduce this mutation and that works relatively well so at the same time that we were doing that work there there has been a lot of groups starting to work on CRISPR systems and people have called that the CRISPR craze and this is just a tiny tiny samples of all the papers that have been published describing the development of CRISPR tools for genome editing in a very broad range of organisms the global idea of all all
these tool work is in all cases you start with programming caste 9 to cut at a specific position in the genome that you want to modify and once you generates this double strand break then the cell can deal with it in different ways so either you can provide a template for auguste recombination and that's where you can control maybe the exact point mutation you want to produce the insertion or deletions etc if you don't provide a template for recombination you can maybe rely on endogenous repair systems of the cell such as non-homologous end joining and HJ and in that case a cell is about to just join together the broken ends but it frequently makes mistakes doing so and like this you can introduce small Intel's at the target position and that's used a lot to introduce knockouts and the last possible outcome is if the cell is not able to repair the break then this will of course lead to the death of the cells and when you look at the country literature so the picture that we have is that this basically to persuade seem to work pretty well in eukaryotic systems actually very well in eukaryotic system to the point that the technology has spread the super fast and now CRISPR editing has become really a standard technology to introduce mutation in eukaryotic systems and in bacteria it looks like CRISPR system is actually pretty good at killing the cells and you can still use that for editing purpose but more as a selection tool than a way to trigger the introduction of a specific mutation and the the fact that
you can actually use CRISPR to kill the cell you can think that maybe you can even use that as an antimicrobial strategy and like the very basic experiments you can do is if you will have a plasmid carrying caste 9 and a CRISPR that you program for instance here to target a kanamycin resistance gene so that's work done in Staphylococcus aureus that plasmid you can transform it very well in the cell the target is not in the chromosome but if the target is in the chromosome then you recover very little transform ants and the idea that the CRISPR is actually going to kill the cells like this we can specifically kill bacteria that carry for instance antibiotic resistance genes or virulence genes of course you need to
be able to deliver the system to all population of cells and the way we're doing that is by using phage as a vector so so phage can are naturally able to to package not only their own DNA but other DNA presence in the cell and that's called transduction and it turns out you can actually increase the frequency of transaction really a lot by simply cloning for instance of packaging signal present on the phage G and if you put that on the plasmids and you can have that plasmid being packaged at a very high frequency in the phage capsid and then you can use this system to inject a plasmid in old population of cells so we're using this this strategy to inject a CRISPR system consisting of cast 9 and a CRISPR that we programmed to target an antibiotic resistance gene present in the chromosome of Staphylococcus aureus cell and the idea is that this should kill the bacteria so this is just to to show you the specificity of it so what you see is loans of cell on a plate and it's actually either a strain that carries academics and resistance gene in the chromosome or that does not and then we program the CRISPR either two targets at kanamycin resistance gene or with a spacer that doesn't target anything and then we just put a drop of our CRISPR phage mid preparation on top of the bacteria alone and we see that it's clearings alone only when the CRISPR is programmed to targets the gene and the gene is present in the chromosome so this is a very specific antimicrobial so you might
wonder why do we want to make anti microbials that are so specific what's what's the purpose of this and the idea is that if you're able to specifically eliminates strains that carry antibiotic resistance for instance then you can take advantage of the competition with other strains that do not carry this resistance gene to more effectively eliminates the threat and to demonstrate this idea we did this very simple experiment where we just put make a culture of two staphylococci strain one that is resistant kanamycin and as this APH genes in the genome and another that sensitive to canonize you know and what we did too is that we put a GFP plasmid in the kanamycin resistant so that we can easily follow both population in you know culture and so this is what happens in the control experiments so the cells are mixed one to one we follow both the optical density and the dashed line is the fluorescence in the culture and so in the control experiment you see that about alfe of the population is the kanamycin resistant cells now what could happen if you use for instance kanamycin as an antibiotic so if you had an infection like that would be a very bad antibiotic choice and here what you see that of course you kill all the sensitive ones and you will only end up with a fluorescent Staphylococcus I if you make a slightly better antibiotic choice so for instance trapped nice in here what happens is that you basically will kill both populations of cells but after a little while you would select resistance in both population and you would still end up with about alphas of population being the bad guys but now if you actually treat with a CRISPR system that specifically target the kanamycin resistance gene so that the purple lines you see that the fluorescent signal here is not recovered so that doesn't mean that we killed all the kanamycin resistance cells but what it means is that we killed enough of them and we let the other population grow in the culture you see that by following the OD curve and this cells not occupy the niche and prevent any possible survivors from coming again so in this specific scenario using a crisper and microbial can actually be more efficient than using antibiotic treatments to eliminate bad bugs so we
showed that we can not only do that in a laboratory strain of staphylococcus aureus but also with some really pathogenic and problematic strains like as a medicine resistant USS hundred strains that are predict from in the US right now and we show that if we make a mixed population with this Merces trend and some non-pathogenic staphylococci we can specifically eliminate the the Mercer strains by targeting the MEK a resistant resistance gene so I also want
to mention that there is actually a very interesting side effect so to speak to this of this strategy which is we can when we treat a population of bacteria we inject the CRISPR system not only in the bad bugs but also in bacteria of the same species that might not carry the target and those are going to survive and if a CRISPR is carried on a replicative plasmids are going to keep that CRISPR and no they are basically immune to always intelligent transfer of the targeted genes so in this case we saw for instance that we can immunize a population of stuff I against the acquisition of the Tri cycling resistance by injecting this CRISPR system and then we show that if we control that as a basically a nonworking CRISPR system we can do a transduction experiment to recover to try a second resistance but if we immunize we cannot do it so that's that's also an interesting possibility
so we went not to do also some animal experiments with this with this ID and this is a skin colonization model in the mice what we do is we shave the back of the mice and then we colonized with a mixture of resistant and non resistant Staphylococcus ions and we treat with our failure mid preparation that target specifically the kanamycin resistant one and so the resistant one our also here a fluorescent so we can nicely follow them after treatment and here we're sure that we're able to specifically be colonized antibiotic resistant bug so it's not as efficient as in vitro but it also works in a more complex environment like the skin I also want to mention that this work is not being pursued by a startup company called illegal bioscience of which I'm a co-founder and that's what seeded Z the institut pasteur so
basically we add this this this picture right now we're cast nine is very useful to make genome editing in eukaryotes in bacteria it tends to rather kill the bacteria and you could asks a question why and is this really always the case will CRISPR really always kill the bacteria and why does it kill bacteria and not eukaryotes so some experiments that we've done recently to address this question is simply to do a very easy assay where we still have a cast line in the cell and we transform a CRISPR that we programmed to target many different position across the genome of e.coli and then we see what happens we basically just count how many colonies do we get when we do this transformation and this is a result that we got that was very surprising at first because we really expected the CRISPR to kill it at any target position but what we realize is that some target position we don't recover colonies meaning that the CRISPR kill the cells but other positions we actually recover just as many colonies as a non targeting CRISPR control and what we saw so that was interesting is that for some of them the one where I put a star the colonies that we recover actually some pretty small colonies indicating that the bacteria here are pretty stressed and this points to the idea that probably first not all targets are equal some targets are better than others and some target position may be tolerated by e.coli and e-coli might be able to constantly repair the breaks introduced by cast 9 at this at this target and we could confirm this ID by simply repeating this experiment in a wreck a mutant so I think you might not see very well there's a white bar here but that's a control if we don't target anything we of course we cover a lot of transformants but all of those there is no white bar basically because if you're in a mutant of the repair systems of E coli rack a then all these targets are going to kill the cells but that still doesn't tell us why does it kill as a cell when when when it actually does and the idea is that it probably does so because when CRISPR is going to cut in the genome is going to cut all copies of of the genome at the same position at the same time and it turns out that most bacteria strictly relying on homologous recombination to repair breaks so if you don't have a sister chromosome to do a Malgosia combination with then basically you're dead and we believe that this is a kind of damage that that Christopher does and to demonstrate this what we did is very simply putting a template for recommendation on a plasmid that is not going to be cut by the CRISPR and the idea is that before I plotted this is right this should rescue the cells and then when we transform or CRISPR system that targets is this like the gene here what we see is that without the repair template we do see a lot of deaths when we when we target the gene but no way if we add a repair template we see that we rescue a lot of the cells so this this confirms that the cells die because they don't have a template for repair then what we we also noticed is that whenever we transform this CRISPR system to target in the position whether it kills or not we were wondering if this could trigger mutation at the targets and because when it kills it usually does not kill all the bacteria you might have some that survive and this is the case for this like the two target here where you have some bacteria surviving but here when we played this on Exile plate so that allows us to see the like the gene is intact or not we see that we recover about half blue colonies and half white colonies so that suggests that the CRISPR actually led to the introduction of mutation at this target position and when we checked what kind of mutation we obtained what we see is that we obtained a lot of very large deletions around the targets deletions up to about 40 Kb and in most cases what was the interesting is that these deletions involve this rep elements of e.coli so I had no idea what these repellents were before I we studied this mutants but those are basically repeat elements so there are a lot of them in the genome of e.coli I don't remember the exact number between 200 and 300 scattered throughout the genome and basically the idea is that they have enough homology between between them that if you introduce the double strand break they can recombine together to repair the break and the cells might survive if of course there were no essential genes in the deleted region so then you can try to make that
but it is why why do you care it excels survive CRISPR breaks so much better than bacteria and one idea you could have is that okay bacteria most bacteria don't have non-homologous end joining but eukaryotic cells do so maybe they are able to repair the cache line break with an HJ and that allows them to survive then we said okay so let's try to introduce an NHS system in e.coli and see if that can rescue the cells and so that's what we did and we took the NHS system from Mycobacterium tuberculosis so I told you that most bacteria don't have an HCG actually there are a few bacterial species that do and and then we repeat the same experiment where we transform a CRISPR system that's going to cut in the in the chromosome so in the control that doesn't target anything we recover a lot of bacteria but here whether we have or we don't have the CRISPR system with the CRISPR system still kills most of the bacteria so it does stick in most of the bacteria but what we noticed is that when we played on this x-gal plate that allows us to see if there was a mutation introducing like Z we see that we start to recover more white colonies with the nhd systems and without and if we PCR the target position we start to see small variation in size at the target positions that are very typical of repairs made by an HJ
and so we can map this dismal deletion and we see that when we have an HJ present in the cell we obtain a lot of deletion ranging from six bases to about 300 bases that are very variable in size but that are still very different from the big deletions we obtain with that energy in the cells and some things that was interesting is that the deletions were always very variable on one side but very always within basically three bases on the other side and that's basically led to the ad that probably cast 9my remain bound more strongly to this end of the break and might protect it from nucleases which might not be the case of the other side explaining the asymmetry in the repair so to sum up this this part basically casts nine when you introduce in bacteria if it's able to cut all copies of the chromosome at the same position at the same time and send the cells cannot do Malgus combination it's going to die but some bacteria might be able to survive by making for instance big deletions and the other things that we found out is that at least with the setup we had nhej a is not able to rescue to rescue the cells it can make some repairs with very very low efficiency and the efficiency is basically even too low to be able to use it really as a tool to introduce in sales in in bacteria so now I want to talk about slightly a different thing you can do with this CRISPR system which is to use the catalytic dead mutants of the Cassadine protein and that's a very interesting mutant because it's still able to find its target position and bind to it very strongly but it doesn't cut anymore and thing is that it by strongly enough for instance to block transcription and so that you can use it to silence Gina so that's something that that we demonstrated the group of sunlight she also a published very similar thing to this to this work and what you see here is just a GFP reporter gene and this is of your relative fluorescence measured and then it's just a position we target with these guide RNAs either within the gene or in the promoter of the gene and the fluorescent level we obtain so you see that if you target in the promoter sequence you obtain very true repression in some cases really barely detectable expression of GFP if you target inside of the gene you can still get very strong repression up to a hundred full repression but then it really depends on the orientation of the of the target in the gene if you target the coding strand you can get good repression if you target the template strain you can only have a much weaker repression and to understand what's happening here you can do a simple northern blot to see this is a base in the control the full-length transcript of the GFP if you target the promoter region you don't see any transcript so basically you block the initiation of transcription if you target within the gene but in the wrong orientation you start to see a smaller transcript here appearing that the size of that transcript matches exactly what you would expect if the transcript is just stopped as a cast non-binding position and if you target now the coding strand here you don't see any more the full and transcript so you have variable repression and you produce this stroller transcript here so basically this decays nine protein Isabel I got to block initiation of transcription or to block even the elongation of transcription and it can be very easily reprogrammed to bind any place you want in the genome so you can use that to silence genes in a very convenient way then you can start to do even more things with the gas 9 you can fuse protein domains to it to introduce functions at a specific position in the genome and here for instance what we did is to fuse it with the Omega subunit of the RNA polymerase and in order to turn it into a transcription activator and then we targeted to bind upstream of weak promoter sequence and we should see activation of the downstream gene and so we targeted it to a different position upstream of the promoter either on one strand or another strand and we see that at a very specific distance from the promoter and in the right orientation we can get up to 23 full activation of of the GFP so I also just want to mention
work by other groups related to to these very interesting applications of the gas 9 for instance including in in imaging if you choose a GFP to dicuss 9 and here for instance it targeted it to bind to telomere sequences you can see here in this picture every little dot is a different telomere in the cells and here you don't have to fix this every single satellite so you can follow the dynamics of different loci in the cell and it's a very powerful tool for people studying the architecture chromosome organization and dynamics so the last application I want to talk about is something that we're really focusing at the moment is to develop screens functional screens based on CRISPR and this comes from the fact that you can so easily reprogram this CRISPR system that you can construct libraries of CRISPR of guide RNAs that are going for instance to target up to 10 to the 5 position in here in the genome of e.coli and then we can introduce this library of guide RNAs in in our cells and then you can perform a functional assay of interest you can may be interested in studying sub MIT antibody concentration some different stresses you may want to combine it with some query gene knockout etc and the readout of this experiment is going to be done through deep sequencing what you do is you sequence the CRISPR library goes before you performs experiments and after you perform the experiment and by comparing or the proportion of each guide RNA changes during the course of the evolution you can basically come to the thickness of each mutant in the population and in series there are two ways you can you can try to perform this this CRISPR screens you can either think of doing them using cast nine to introduce knockouts in combination with in HJ and that's something that people have already published and are doing in eukaryotic systems where you have this endogenous and HCG in the cells and these screens work quite very well and you can also think about doing screens based on de caste 9 where here you're going not to introduce knockouts but to knock down and silence the target the target position and I explained to you that an HJ repair at least in e.coli is very inefficient so we cannot do this but we can do this type of screens but ideally we might be interested in both and both of type of screens have actually different properties that might be complementary so for decades nine screen you can see that you would only have partial silencing and pull our effect what you actually know what the polite effects are for instance you know Paran if you knock down targeted gene early into around it will also block expression of all the downstream genes a big advantage of dicas nine Odom's is that it's actually boost inducible and reversible so knockouts with cast nine and hgj here the good thing is that if you really have a frameshift you can hope to have complete deletion but the problem is that the elites male are going to be quite unpredictable and you don't really understand what the polar effects will be I just want to give you a flavor of what kind of data we can get with this type of screen and this is just a very raw data showing you the number of reads we get for each guide in the library both before the experiment and after the experiment so this is a controller we don't induce the expression of the gas 9 so everything falls more or less nicely along the diagonal meaning that the number of guidance the library didn't change between before and afters experiment but now if you start to induce the expression of the gas 9 you see a lot of points either going down opponent is going up and power is going down here means that basically those guys provide a fitness defect to the cells in the population and guys going up we provide a fitness advantage to guys in the population and so then you can of course
focus on some specific points and try to see what what's going on and here for instance is the exact same figure except that I just kept the targets in this mirror aging that's an essential gene in e.coli involved in cell wall synthesis and what you see that the control experiment everything is still nicely on the diagonal but no when you induce you see that here's also these pink points basically go down and the green one blue one stay stays there and what this points highlight is these blue points actually target the template strand and if you remember from a food qslide before I explained that targeting the template strand gets you only very weak repression but if you target the coding strands with that those are these red points here you have very good repression and use the depletion of this guide in the library which is expected because if it's an essential gene so like this we can very easily basically figure out what are all the essential genes in the cell and of course if you do that in a specific condition where you want to do from channel screens you can also get more in the interesting information something that we think is also very interesting and that's linked to the fact that this is something that you can induce at a precise moment you can actually follow the evolution of of each guide over time and here we sequence for instance this library after 20 generation of culture or after 40 generation of culture and this is target's in the mercy or sec D gene so here I kept the same color code the blue ones target the coding strength so good repression so red one target template strain weak repression so you see that with all the red lines are basically straight horizontal lines and the blue lines here both these rings are essential genes but it turns if you target mercy the target the guys are depleted much much faster in the population then if you target SEC D so both genes are essential but maybe you can start to see some degrees in essentiality if you want to say so where
we're going with this basically is that this single knock down knock down or knock out screens actually very cool and in some ways actually also pretty similar to what people have been doing already in the past for instance with transposon tag meta Jenny's TN seek kind of approaches but what we see we can do that very exciting with this is now go after generating interaction by doing multiple knockdown simultaneously in the cells and so that's that's some things that that were really excited in doing right now and we're just starting to be able to to construct these double knockdown screens and study them in the same way so with this I'd like to to
thank people in my group so we have a very nice lab space at the institut pasteur we just started a year and a half ago and not to my knowledge it would be super easy to check whether Deinococcus I tend to add or not CRISPR system it turns out that something like 50 percent of bacteria of CRISPR systems so there is this very nice database CRISPR database online that we could look into I never checked for dinner cause III don't know a few slides back in your mercy data there are different generation times some of the of the lines on the graph point up 40 generations is that just an artifact of way of measuring or assess some recoveries so we're not quite sure yet we're just starting to look into this type of things so the true answer is I don't know I suspect that it might actually be some mutant guide RNAs that might survive and then have a regular fitness or recover beat or something I but III honestly I'm not quite sure so you showed that the crystals killing these cells unless you provide the template for repair if there's whatever yes which means - yeah so you putting your finger on very very good points and the answers that we first believed so as well and it actually turns out not to be the case and we were lucky to have this experiment where actually the template is is able to rescue the cells efficiently but it turns out that this works only if you have if what you introduced is a point mutation and and only for a specific ology learn the lens of your mammalogy arms to need to be very specific and as soon as you want for instance introduce a small initial efficiency drops dramatically and etc so we're and we don't understand really why yet and this is something that we're investigating as well I think I'm just not quite understanding first screen so it's similar to TMC transposon week in some way so when I was wondering compare and contrast in that business the second is how are you preventing death in this case rights issues so here it's using the D cash nine so we're not cutting the genome it's just going to silence expression of the target positions so we're not killing and then but you're perfectly right that it's very interesting to compare this type of method with T and sick and we're actually doing that at the moment so we're conducting in parallel and we're going to so so you can design them so now you have these companies including your I think probably the one that is most commonly used is old ego arrays so I mean I don't have any charge enzyme whatsoever I'm just think the ones that we're using they provide as you can order a pool of all ego and you can precisely determine the sequence of up to ten to the five different only goes you want to be in this pool and they just generated for you so the technologies that they're using are very similar to the technology used to generate microarrays except that the ended is just a cleave it off the chip and recover it in a pool and you get that and then you can leave at this pool of all the goals that you just use it in a standard cloning procedure so and what's the role of the pen sequence you shot this one promoter will get nicely spaced target are nice so you absolutely need a pan motif to be able to to cast nine to bind so casting is really first looking for the penalty if there is no penalties it's not going to bind and so in the experiments where I show that so for instance we had we tested many different positions along with promoter we specifically engineer the promoter to contain many Pam motifs so that we can test precisely different positions so would that be a problem if you want to target a gene and then you so the answer to that is is that you actually quite you have quite a broad range of position you can target even if what you want to block is the initiation of transcription you see here it's a hundred base pair range where you are still very good blocking the initiation so GG motif is very frequent it's been randomly every eight pages so sorry I'm just wondering and you were talking about you immunizing with the CRISPR when you're treating population to kill the bad guys so how long does that musician last so it should last as long as the cells keep the CRISPR system then along will the keep it will depend on as the ability of the plasmids that you inject and many other things so that we haven't really investigated the cells the sounds were I'm not sure I understand which cells are talking about well previous question about what's about the period of the existence of resistance and you refer this period to the cell survival so cells how long do they survive in your system I mean if they're not killed by the CRISPR systems are going to survive indefinitely there's barely one couch over here mr. question about the natural or CRISPR system aha how many repeats natural bacteria maintain so that's very very very variable it's basically from one to six hundred and depends on different bacterial species and yeah yeah so and it's this is really not understood why some species of bacteria tend to have shorter CRISPR where's on a larger CRISPR arrays yeah I cannot really speak to that it's really a still a mystery