Enciclopedia - Page #1169

Parte 5 | Gabriel Lamothe | Seminário Internacional de Ciências Farmacêuticas | Método CRISPR VI

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0:28 welcome thank you for having me uh let's see if 0:34 we can get it started hi my name is gabriel lamot and today 0:41 i'll be discussing the detection of cyrus kobe yeah that's much better is the sound 0:47 better now b2 using crispr technology oh perfect namely the test that we've been developing in dr tremblay's laboratory 0:55 studies have confirmed that many individuals infected with the sars kobe 2 virus are asymptomatic carriers of the virus 1:02 this is problematic in that the pandemic might be growing without adequate supervision simply focusing the testing on 1:08 individuals with symptoms as opposed to entire populations is not enough 1:14 to better contain the virus until a large scale distribution of an effective vaccine is underway large scale and 1:20 continuous testing is mandatory the current diagnostic pipeline for 1:26 detecting sars cov2 is a three-step process the first step is to obtain nasopharyngeal swabs from 1:32 patients the next step is to isolate the total rna from said samples and provide the rna required for this 1:40 third step which is to use real-time quantitative vcr machines the rna is first reverse transcribed 1:46 into dna and then amplified a dye is added to the amplification reaction to signal the presence of dna 1:52 strands after a certain number of amplification cycles a positive result will give 1:57 a strong enough signal that the machine flags it as containing the original virus now this process is problematic for a 2:04 few reasons the first is that real-time pcr machines are extremely expensive 2:10 most can range from 15 000 us dollars to well over 90 000 us dollars 2:16 given that it's a fairly specialized piece of equipment and that not all molecular biology laboratories need one 2:22 some get along just fine with a regular pcr machine which costs a much more reasonable 5000 us dollars 2:28 as such widespread testing for the coronifiers using these machines isn't ideal 2:33 acquiring the large number of machines required for widespread testing is a feat in and of itself 2:39 and is likely one of the contributing factors for the sometimes long wait times for results 2:45 many reports have stated that it can take 3 to 24 hours or more to obtain results in a clinical 2:51 diagnostic laboratory over here in canada ontario reports wait times of up to four days to get results 2:58 high-density metropolitan areas like new york are also subject to long lines for testing and then a time to 3:05 results of about seven days for some clinics in that city the creation of new types of detection tests 3:12 is therefore an important step in curbing the spread of this virus an ideal test needs to follow the world 3:19 health organization's criteria for assured that is the test needs to be affordable 3:25 for as many people at risk of infection as possible tests that require extremely expensive reagents or machinery 3:31 are not ideal the test must also be specific to avoid false negatives that way anyone 3:37 who could potentially spread the virus can be identified and put into quarantine the test must be sensitive 3:43 so that few people who don't have the virus are mislabeled as having it and treated improperly 3:48 the test must also be user friendly that is it must be simple to perform and it must be rapid and robust that is 3:55 it must give a time to results that is quick enough to make the most use from the results situations where it can take up to seven 4:02 days to obtain results are clearly far from ideal as by the time the patient receives 4:07 the information they might have infected many others the test must be equipment free as much 4:13 as possible tests that require too many rare and expensive machines aren't ideal for many 4:18 countries finally the test must be deliverable to those who need it 4:24 the discovery of cast 13 or c2 c2 was revolutionary in the field of molecular detection 4:31 this class 2 type 6 crispr effector is similar to the protein cast 9 off of 4:36 which base ending and prime ending were designed this protein is also a member of the crispr family 4:43 unlike cas9 however cast 13 can be programmed to target rnas as opposed to dnas 4:49 by binding to its crispr rna cas13 is primed to target extremely specific rna sequences after locating a target 4:57 sequence the cast 13 protein is activated this causes the protein to begin to cleave 5:03 everything its surroundings in an event called collateral cleavage it cleaves its original target 5:08 and then moves on to start cleaving all surrounding rnas researchers realized that this 5:14 collateral cleavage could be used to turn the protein into a switch for a detection test once 5:19 the target molecule is present the switch is flipped the cast cas13 activated and all surrounding rnas are degraded by 5:27 tagging certain rnas in the solution with a fluorescent molecule and a quencher 5:32 it would therefore become possible to resolve a signal researchers therefore started to develop 5:38 a detection test based on this protein for viruses bacteria and human diseases such as cancer 5:45 however they soon noted that there were certain limitations to the protein and that is limited detection was far from sensitive 5:51 enough for their needs recently certain tests have been designed using only cast 13 5:57 however these tests are less sensitive and tend to use a different cast 13 protein 6:03 researchers at the broad institute decided to supplement the protein with a few others that would amplify the 6:08 target rna to this end dr zhang and his team created a new test called specific high sensitivity 6:16 enzymatic reporter unlocking or sherlock for short this test is based 6:21 on the reverse transcription of target rna into dna afterwards the dna is amplified at a 6:27 constant temperature by using a process called recombinase polymerase amplification 6:32 this technique amplifies a small amount of dna at a constant temperature unlike the more commonly used bcr 6:40 the incorporation of this step also means that the ensuing detection test can target dna since the rpa reaction 6:47 simply amplifies a determined section of dna using two primers the initial genetic sequence can either 6:53 be rna or dna so long as reverse transcriptase enzymes are present for rna 7:00 the amplified sequences are then transcribed using a t7 rna polymerase to create vast quantities of the target 7:07 rna this then allows the cas13 to have a far larger pool of target molecules 7:12 which can activate the enzyme once the enzyme is active it does what we discussed before 7:18 and starts to cleave all surrounding rnas including reporter rnas which give off the easily detectable signal 7:24 which makes the test work in their initial version of the sherlock test two different reporter 7:31 molecules were used the first used a fluorescent molecule as well as a quencher 7:37 and it was able to produce a lot of fluorescence after cleavage of the rna the other 7:44 was designed to interact with a few antibodies and when it was cleaved it appeared as a distinct 7:50 second band on a strip that was quite similar to a pregnancy test 7:55 while this test was good in many ways it had certain drawbacks at the beginning of the pandemic we attempted to replicate it and 8:01 immediately had a few issues the biggest issue by far was that some of the materials required for the test 8:07 to work were only available in extremely limited supply the kits required to perform rpa 8:12 reactions for example took approximately two months to arrive and even then we only received enough 8:18 materials to perform 200 tests this clearly wasn't going to be enough to produce sufficient tests in the 8:24 future additionally the reporter that used a pregnancy style strip was remarkable and seemed to be very 8:31 useful however since it was too difficult to acquire insufficient quantities 8:36 we decided not to use it and to instead focus our efforts on using the fluorescent style reporter rna since the original version 8:44 of the test could not be used by us in the way that it was intended we decided to redesign it 8:51 in redesigning the test we had several options and routes that we could take we knew that we wanted to keep on using 8:57 cast 13 because we had a large supply of this enzyme due to an ongoing collaboration with dr 9:02 alain ghani's lab we also knew that we wanted at all costs to maintain the isothermal nature of 9:08 this test one of the biggest advantages to sherlock was that it didn't require large amounts of expensive equipment 9:14 simply by using a heating block or something of the sort it was possible to get a visual readout 9:20 we therefore decided to employ loop-mediated isothermal amplification or lamp for short this amplification 9:27 strategy was fairly well documented and easy enough to perform based on the use of six primers this 9:33 technique could rapidly amplify large amounts of target dna unlike both rpa and pcr however 9:39 lamp doesn't create identical amplicons of a discrete size instead the six primers can interact to 9:46 create far larger molecules of a repeating sequence initially it was a little difficult to 9:52 incorporate this isothermal amplification strategy into our test as you might remember the step following 9:58 rpa in the original sherlock test was one that produced lots and lots of target rna 10:03 this production of target rna was only possible because the rpa step added a tag that let the t7 rna 10:10 polymerase bind to the dna and begin transcribing it however no one had seemingly ever done 10:16 that with lamp and since this new technique used six primers two of which looped back on themselves to create 10:22 weird dumbbell like structure structures it was difficult to figure out exactly where to incorporate this tag 10:29 after a while we decided to include the tag right in the middle of one of our looping primers this has consistently 10:35 given us very strong results we were therefore able to retrofit the original sherlock 10:41 test with a new amplification strategy all the while still benefiting from cast 13's specificity and signaling 10:48 the new test is therefore using rt lamp as opposed to rt rpa of course 10:54 should we become interested in targeting dna in the future for either certain types of viruses or 11:00 certain bacteria this test would be readily applicable to that as well we also decided to use a fluorescent 11:07 reporter rna instead of the pregnancy style one that we spoke about previously because it seemed to be the most readily 11:13 available material since it doesn't take any particularly advanced machinery to make it work 11:18 we were happy to choose this one 11:24 now that you know how the test works from a theoretical standpoint let's discuss it from a more practical point of view for now 11:31 the test requires that we start with rna that was extracted from a virus using a commercially available kit one 11:37 microliter of this solution is transferred over to the tube containing the rt lamp reaction 11:42 ideally in the future we would like to modify the test so that it becomes possible to directly add a patient's saliva 11:48 to it and continue on from there that would noticeably cut down on the labor required to obtain each reading 11:54 cut down on the overall cost and make it much faster for the results to come in 11:59 after transferring one microliter of the rna extract to the first tube the tube must then be incubated at 65 12:05 degrees celsius for 30 minutes after that one microliter is transferred from the first tube 12:11 into the second which contains the t7 and cast 13 solution after tapping the tube a few times to 12:18 mix it and microcentrifuge it down for a few seconds all you need to do is incubate that tube at 37 degrees celsius 12:25 for 30 minutes at that point all of the reactions are done and that the results are ready to be 12:31 read by exposing the tubes to a wavelength of approximately 490 nanometers 12:36 it's possible to easily detect which samples contain cyrus cov2 those ones will fluoresce a bright green 12:43 those that didn't contain any of the viral specimens of interest will be completely colorless and indistinguishable from a tube filled 12:50 with a little water i'll show you that later now it's worth noting that the dna and rna sequences 12:57 that we have used in this first version of our test were all previously published by different groups 13:03 as such we do not believe that they will result in a positive signal when in the presence of a relevant rna 13:10 this is particularly important because your saliva is full of rna the extraction process for the virus 13:16 does indeed indeed result in viral rnas being captured but there tends to be a whole lot more 13:22 human and bacterial rnas too in addition other coronaviruses that are 13:27 much less dangerous are fairly common in the human population if our tests gave positive signals every time someone came in 13:34 with a common cold then it wouldn't be very effective as such we have based ourselves on 13:39 previously described templates to try and avoid this as much as possible 13:45 in designing this test we tried to make sure that it wasn't overly reliant on complicated machines 13:50 we wanted the entire test to be easy to perform using only a few common laboratory apparatuses 13:56 as a result with this current iteration of the test all we need is one machine that can heat 14:02 pcr-style tubes to 65 degrees celsius and one that can heat 14:07 the pcr tubes to 37 degrees celsius we also need two either p2 or p10 14:14 pipettes for reasons we'll get into later along with their associated peptide tips 14:20 a small microcentrifuge capable of taking pcr tubes is also required just to make sure that the reaction 14:26 mixtures don't touch the top of the tube when you're opening them and potentially contaminating 14:31 all of the surrounding tubes finally a machine capable of giving off ultraviolet wavelengths is necessary 14:38 in this picture you'll see the machine i've been using it's most commonly used to visualize gels in a molecular biology 14:44 lab but it does a really good job of visualizing our positive samples in our test for the two incubation 14:52 periods several different options are available i've been using an old thermocycler today 14:57 as i find it does a really good job of evenly heating my tubes it's worth noting here that this means 15:03 that even old and run-down thermocyclers that no longer do a very good job of cycling between temperatures 15:09 can be readily used in this test unlike the current rtq pcr gold standard that requires 15:16 specialized real-time thermocyclers here any old machine can be used 15:22 it's also possible to use other common laboratory apparatuses such as block heaters that have an 15:27 adapter for pcr tubes please note that in this picture the block heater has an adapter for 1.5 15:34 millimeter microcentrifuge tubes so the true adapter will look a little different 15:39 i've also used incubators to great success even bacterial incubators that operate at a steady 37 degrees 15:46 celsius can perfectly suit the needs of this test so long as the tubes are readily exposed 15:51 to the ambient temperature then positive signals should have no difficulty in resolving 15:56 please note however that when using incubators it's better to have the tubes exposed to the air 16:02 that way they don't waste time as their rack slowly begins to heat up 16:07 water baths are one final option that you can choose personally i tend to avoid this option 16:13 because i find the risk for cross-contamination between samples to be much greater and therefore unideal 16:19 however i have tried to perform rt lamp reactions with this system and so long as you're 16:24 careful it's very possible to do it 16:30 once the reactions have been completed it's possible to use various different machines to visualize the tubes depending on the 16:36 materials your laboratory has personally my machine of choice is a gel uv emitter 16:42 these machines which are more commonly used to visualize agarose gels do a very good job of safely showing 16:48 which tubes are fluorescent and which aren't black lights can also be used 16:53 for this test however i have found them to be slightly less desirable since the reporter rna best absorbs 16:59 wavelengths of 490 nanometers i found that black lights which produce wavelengths of 390 nanometers 17:06 to be less favorable for this test for them to work it's better to visualize the tubes in a dark room 17:13 in both cases the machine should really be placed in a darker room away from excessive light a little 17:19 background light seems to have less of an effect in the case of the gel uv light however 17:25 this picture seen on the left was taken using a generic iphone when the tubes were placed on the gel uv 17:31 light the lights in the room were only partially turned off resulting in lots of ambient lighting 17:37 even if there were no lights directly pointed at the machine given the strong and even radiation that the tubes 17:44 received on this machine it's easy to determine which samples were positive and which were negative 17:50 in the case of the black light on the right it's clear which samples are positive and which are negative 17:55 of course however the fluorescence is not even and it's a little bit harder to detect 18:01 by the naked eye in this case the camera on the iphone took a very good picture 18:06 and made it easier to visualize the tubes overall now there are several important 18:11 considerations to be had when using this test for it to work properly and not give either false positives or false 18:17 negatives it's important to first designate proper working stations for each section of the test it's also 18:24 necessary to follow the predetermined reaction times without varying otherwise the results might start to 18:30 differ slightly finally those handling the test must be careful not to contaminate any of the 18:36 tubes with rnases the first consideration to separate 18:41 working areas is extremely important because lamb is excessively sensitive this technique is known to produce lots 18:48 and lots of concentrated target dna we as well as others have noted that 18:54 when we open tubes that completed the lamp amplification in the same area that we originally used 18:59 to set up the reaction it's possible to contaminate the working area with aerosols containing the 19:04 product if you continue to set up the lamp reactions in those areas afterwards then you run the risk of 19:11 contaminating your new tubes with amplicons from the previous reactions by touching your desk or 19:16 various other contaminated surfaces with your gloves you run the risk of artificially seeding your reaction 19:22 mixtures that contain patient rna with no sarisco v2 and therefore producing positive results 19:29 when there really shouldn't have been any by separating the areas in which you set up the lamp reactions 19:34 and those in which you use the amplified sequences you run less of a risk for this to happen it's 19:40 also necessary to have designated lab coats pipettes and other such materials for each working station 19:47 if those in workstation 2 become contaminated you don't want that coming back into workstation 1. 19:53 please note that this is much less stringent when bringing materials from workstation 1 to workstation 2. 19:59 since it's the rt lamp reaction that runs the greatest risk of contamination bring material for example gloves 20:06 from that station to the next should not be a problem provided that the gloves are still 20:12 relatively clean please note that this situation of contamination via aerosol 20:18 is of much less importance during the second stage of the test as we discussed previously the cast 13 20:24 protein we are using requires a relatively large amount of rna to be functional 20:30 at the present time we are not concerned in the least then aerosol contamination from lamp 20:35 might produce false positives of our cast 13 reactions as we have yet to see a single situation 20:41 where this has happened the second consideration of note is that 20:47 the reaction time must be followed a small discrepancy can be tolerated that is one minute more or less doesn't 20:54 really seem to have a large impact on the test however more than that can start to become problematic 21:01 in the case of the rt lamp reaction external teams as well as us have seen that by doubling the reaction 21:07 time for the lamp false positives can arise as you can see here in both pictures 21:13 a successful lamp reaction can also be visualized on an agarose gel the successful reactions are 21:19 characterized by the presence of several large bands of varying sizes however when the lamp 21:25 reactions go on for too long it's possible to begin amplifying samples with nothing in them 21:30 for example on the left you'll see a gel that was taken from an article in which at 30 minutes the wells 21:37 containing reactions with 10 copies of the target are viral rna and no copies of the 21:42 target viral rna had no bands in the case of the gel on the right a gel that i made three of the wells 21:50 contained reactions that had no target rna to speak of only the n fragment well contained 21:56 target rna at 30 minutes these negative controls did not demonstrate any amplification 22:02 as we expected however at 60 minutes both the published gel and mine started 22:08 to give amplified products in their case the ten and zero copies both started to be amplified 22:15 and in my case one of my negative samples started to get amplified i've performed 22:22 several experiments so far with 30 minute incubation times and so far have never gotten false positives that's 22:29 not to say that it's entirely impossible simply that it's very very unlikely as a 22:35 side note this is a good time to mention that changing gloves frequently when performing the first reaction 22:41 is a good way to avoid these kind of unlikely false positives after prolonged use gloves might become 22:47 contaminated with small amounts of viral rna and that could potentially lead to false positives even if we're only incubating 22:54 at 30 minutes the final consideration to look out for 22:59 is the presence of rnases for those of you who might not be familiar rnases are enzymes that cleave 23:05 all rnas they come across both steps for this test are sensitive to rnases 23:11 the initial arty lamp begins its amplification off of rna so the presence of rnases could be very 23:17 detrimental and therefore lead to false negatives the second step of the test the t7 and cast 13 step uses 23:25 an rna reporter to signal positive tubes if while you're using the tests you start to introduce rnases 23:31 then you run the risk of getting false positives rnases are extremely difficult to remove 23:37 once they're introduced into a system so the best means of protecting your test is by using prevention 23:43 since your hands produce lots of rnases it's mandatory to use gloves at all times when working with the test 23:49 in doing so you will prevent your body's natural rnases from degrading the initial star's cov2 23:54 rna and protect the fluorescent reporter at the end gloves must also be changed whenever 24:00 they come into contact with skin hair or other frequently manipulated objects 24:06 such as doorknobs and personal devices the use of rnas and dnase free 24:12 filtered pipettes is also important for this test by using these kinds of specialized tips 24:19 you can make sure that any possible contaminations you might have in your pipettes doesn't have a chance 24:24 to contaminate your test in areas where the risk for rnas contamination is fairly high 24:30 it might be worthwhile to treat all surfaces or tools with an rnas removal solution these 24:36 kinds of solutions can degrade rnases that might otherwise stick to your gloves and contaminate your samples 24:42 please note i haven't had to use the solution very often so long as you maintain a clean 24:48 working schedule you should be fine the second component for the test 24:54 the one containing t7 and cast 13 is also designed to be able to tolerate a 24:59 certain level of contaminating rnases there is an rnas inhibitor added to the reaction mixture 25:06 to prevent the degradation of the reporter in case the original sars kobe 2 rna 25:11 wasn't completely clean or in case contaminations occurred while you were preparing the tests 25:18 that said the presence of excessive rnases can still fully activate the test seen 25:24 here on the right one microliter of very concentrated rnase a is capable of causing the test tube to 25:30 become fluorescent it's therefore important to take the 25:40 required 25:54 cabrillo 26:02 test that can be widely distributed while also meeting this limit of detection is fully functional for curbing the 26:10 spread of this pandemic while rt-qpcr is much more sensitive 26:15 than this its improved limited detection might not necessarily be that beneficial 26:20 since this technique can pick up rnas freed by infected and dead cells after the infection has 26:26 run its course this form of screening can cause individuals who are nominally sars kovi to rna positive but aren't 26:34 actually infectious to be isolated unnecessarily as you can see here on our left our 26:41 detection test is able to detect samples that have 100 copies per microliter as was stipulated by the model a more 26:49 recent comparison between the different primers that we've been using for lamp has demonstrated that one version of our 26:55 test can detect samples containing only 80 copies per microliter while the other can only detect samples 27:01 containing 1 000. moving forward we're likely to focus our efforts on the first primer set to ensure that 27:08 we meet the limit of detection that was stipulated by the model 27:13 as a final note we're happy to report that our test works not only on fragments of the cyrus 27:19 cov2 genome it also works when exposed to the virus's full rna profile 27:25 initially we began the development of our test by using fragments of the viruses genes that we had 27:31 transcribed ourselves in the lab recently we've used the test on actual 27:36 sars cov2 viruses and have obtained the expected results as you can see on the left 27:42 the first section of the reaction the rt lamp reaction works just as expected on the viral rna 27:48 the top left wells that were run on the agarose gel clearly demonstrate a strong amplification below them three 27:56 reactions were performed with cells that had not been cultured with the virus the viral rnas were created by first 28:02 isolating the viruses from a patient these viruses were then cultured in viroe6 cells 28:08 and after a few days the culture medium was treated to extract any and all rnas that were present the 28:15 viruses were therefore destroyed and their rna was obtained when the lab amplicons that had 28:21 amplified the real virus were transferred over to the second stage of the test they gave the expected 28:26 strong fluorescent signals at the moment what remains to be done is to use our tests on samples 28:33 that have been confirmed positive or negative by rtq pcr the current gold standard in sarsko v2 28:40 detection and see how our test compares this will give us a better indication 28:45 of what our tests sensitivity and specificity are just to wrap up 28:52 i would like to thank dr gary cobinger dr ellen gianni and dr ghibwave for having helped to 28:58 provide finances or materials for this project i'd also like to thank dr tremblay and everyone in his team 29:04 for having helped provide certain insights as i was redesigning this test finally i'd like to thank dr marcelo for 29:12 having helped to organize this entire meeting and the canadian institute for health research 29:17 and the frqs for having helped finance me as i pursued this project 29:27 okay so that was my presentation uh yeah 29:34 thank you gabriel uh i'll take the time just to make a uh to summarize it the best i can here 29:41 for the non-english speakers thank you very much for the presentation thank you okay

Parte 4 | Luiz Werber-Bandeira | Seminário Internacional de Ciências Farmacêuticas | Método CRISPR VII

TEXTO DO CANAL BRASIL SEM ALERGIA NO YOUTUBE

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0:00 o ok vocês terem uma ideia da 0:04 importância dessa pesquisa laboratório 0:07 Doutor Jackson André Recebeu agora uma 0:11 bolsa do governo canadense de milhões 0:13 $600000 para para esse pesquisa do 0:18 creeper utilizando na terapia de doenças 0:21 nós estamos trabalhando com ele também 0:25 aqui no Brasil e junto com a procura do 0:27 Rio de Janeiro e nós já estamos 0:30 validando um novo peste é para detecção 0:35 de vidro e poderá ser usado também no 0:41 convívio de 19 esse testes no validado 0:45 quando Laboratórios consciente o 0:47 professor a Fiocruz do Rio de Janeiro e 0:51 ele detecta a presença do RNA viral em 0:55 duas horas e é é um teste akita inu 0:59 preço 1:00 a aparelhagem tocar pode ser feito em 1:04 qualquer lugar dos países tinham no 1:06 interior e e por qualquer pessoa não 1:10 precisa ser um técnico laboratório ele 1:12 pode ser Senado e nós recebemos aqui no 1:15 Brasil para essa peça técnica 300 mil e 1:19 uma parte também veio da terra

Parte 3 | Jacques Tremblay | Seminário Internacional de Ciências Farmacêuticas | Método CRISPR VII

TEXTO DO CANAL BRASIL SEM ALERGIA NO YOUTUBE

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0:00 thank you for the invitation let me start my presentation 0:11 using the crisper cast 9 technology 0:17 today's presentation will be divided into part i will first present the use of the 0:22 crispr cas9 technology to treat iris daily disease and my graduate 0:27 student gabriel de mart will present all the crispr cas9 derived 0:32 technology may be used to detect viral infection 0:39 the word crispr stands for clustered regulatory interspace short penetrate repeats 0:48 crispr cas9 technology is derived from research on bacteria it was initially discovered in 2005 0:56 that the crisper sequence contained viral sequence and yes there was an 1:02 hypothesis that crispr cas9 is the bacterial immune system 1:07 it's only in 2013 that it was discovered that the crispr cas9 1:13 may be used to induce specific modification of the human genome and this started 1:20 a big explosion of artificial indeed the crispr casting system is used 1:28 by bacteria to kill bacteriophage bacteriophages are extremely abundant they're 10 times more 1:34 abundant than bacteria and there is a war that is ongoing 1:39 between bacteria and bacteriophage since millions of years 1:46 it was initially discovered that they are in the genome a constant repeated sequence 1:55 researchers were wondering what these repeated sequences were for and then they discovered that the 2:01 sequence between the constant repeat or sequence that were up from the genome of various 2:08 bacteriophage in fact what normally happens is when a 2:14 bacteriophage infected bacteria normally the bacteriophage wind bacteria is dead 2:20 but sometimes you have a defective virus that will infect without killing the bacterium but they will then 2:26 be able to acquire some part of the sequence of the virus and it will store that into its genome 2:34 and it will be ready when it needs such bacteriophage again in the future 2:41 from the dna in the crispr area bacteria will first express the pre-cr allergy and then there will 2:48 be a complex that will be made between tracker or cr rna 2:53 and the cast iron protein this striker rna crna cas9 protein 3:00 complex will then bind with the bacteria of hdna and induce a cut of the bacteriophage 3:08 dna to essentially kill the virus 3:14 to bind to the dna the cas9 protein requires the presence of the total spacer 3:20 adjacent motif which is simply njj for this streptococcus pyrogene 3:29 then there is a crrna which contain a variable sequence of 20 3:35 nucleotides that will be complementary to the bacteriophage sequence and then there is also the tracker rna 3:42 which is a constant rna sequence that forms a complex with the crm 3:48 when the three parts are together cr rna tracker rna in the cas9 protein 3:54 the cut will be induced at exactly three nucleotides 4:02 of course since this is a war there are a counter-measure that are taken by the phage they will 4:07 mutate their genome so that they become resist and they are not recognized by the cr 4:13 rna and will survive bacterial defense 4:20 the crispr cas9 big bang started in 2012 when gen x and colleagues realized that 4:27 the crispr castling technology can permit to cut the genome of planets and animals they also 4:35 fuse their cr rna and the tracker rna together to form a single guide rna this was done 4:44 essentially because the single guide rna is something that can be patented 4:49 whereas the cr rna and the tracker have any being natural it cannot be patented 4:58 during the following year there was an explosion of article confirming that the crispr technology 5:04 may be used to cut the genome of bacteria human cells carb animal mouse frogs zebrafish plant flights 5:12 limited these monkeys 5:17 many of these articles were published in prestigious journals such as nature 5:23 science nature biotechnology molecular cell cell molecular cell 5:30 there was a rapid explosion of article and as you can see as of november 26 2020 5:38 21 420 articles have been published where the word crispr is mentioned 5:47 the main reason of this rapid explosion of article is that it costs much less to derive 5:54 a sequence that will be able to cut a specific genome sequence compared to other 6:01 pre-existing technology such as zinc finger protein and tailors 6:07 as mentioned before the binding of the cas9 protein to the dna 6:13 required the presence of a bottle spacer adjacent concept pam which is njj for the cas9 6:20 derived from skeptical g there is then the binding also of the 6:26 single guide rna which will recognize the sequence of 20 nucleotides 6:31 and when the complex is made between the cas9 protein the single guide rna the dna there 6:38 is a cut that will be made at exactly three nucleotides from the pan 6:46 once inside the nucleus the resulting complex will lock onto a short sequence known as the pan 6:54 the cas9 will unzip the dna and match it to its target rna 7:01 if the match is complete the cast 9 will use two tiny molecular scissors to cut the 7:06 dna when this happens the cell tries to 7:12 repair the cut but the repair process is error-prone leading to mutations that can disable 7:18 the gene allowing researchers to understand its function [Music] 7:23 these mutations are random but sometimes researchers need to be more precise for example by replacing a mutant gene 7:30 with a healthy copy this can be done by adding another piece of dna that carries the desired sequence 7:38 once the crispr system has made a cut this dna template can pair up with the cut ends 7:43 recombining and replacing the original sequence with the new version [Music] 7:53 one of the reasons of the rapid explosion is that increased bypass like technology also used all what was developed by gene 8:01 therapy in the 30 years that preceded it we have better to delivering the dna 8:07 using aav associated virus cationic polymers liposome 8:12 microinjection electro operation all of those techniques are used in the context of crispr 8:20 when a double strand break is produced in the dna most of the breaks will be repaired by 8:26 non-homologous adjoining that will lead to micro deletion or micro 8:32 insertion this is frequently used to knock out the expression of a gene by 8:37 changing the reading frame and it can also be repaired by homology directed repair 8:43 which require the presence of a donor dna that contains sequence of homology with what 8:49 is receding what is following the cut and in between the two homology sequence 8:55 there is a sequence in blue which may be a single nucleotide or a whole gene that will be 9:00 inserted at the side of the double strand break 9:06 when we use the crispr cas9 technology we intend to produce a double strand break 9:12 at a precise site in the human genome however the cas9 may sometimes induce cut at 9:18 other sites in the genome which are called off-target mutation computer software may predict these 9:25 off-target mutation by using the whole human genome sequence however 9:30 these softwares are not perfect and they will sometimes print it off off-target 9:36 mutation at site where there is no cut and sometimes there will be a cut at the side which is not predicted 9:42 by the computer there are or however some experimental 9:50 techniques such as a guide sec method which permit to experimentally identify the sites 9:57 of target mutation this guidesec technique used the introduction of a short 34 base 10:05 pair oligonucleotide at the site of the cuts and later by pcr 10:10 the site of insertion may be identified by sequencing 10:16 the presence of these off-target mutations is the main problem that delays the use 10:23 of crispr cat9 technology for direct vehicle corrections 10:31 one of the method to reduce of target mutation is simply to reduce by two or three the 10:37 number of nucleotides of the single guide rna which is binding with the target dna 10:46 another method is to use a mutated cas9 nuclease that cut only one strand of dna it is 10:53 then called the kneecase there is variation of cas9 where the nikkei cut only the lower 11:01 strand of dna and other mutation of cas9 which permit to cut only the upper strand 11:08 of dna it is just possible to induce more precise 11:14 double strand break in the dna by using the cas9 nikkei 11:19 and two single guide rna each detecting a sequence of 20 nucleotides close to 11:25 one another it is also possible to use a 11:30 non-functional cast 9 that then cast 9 nuclease fuse 11:36 with the one nucleates then two guide iron area needed to detect 11:42 sequence which are close to another and the front one nucleas need to form a dimer 11:48 in order to be able to cut the dna 11:53 finally some researchers have mutated the gene coding from cas9 they have 12:00 modified some sequence coding for amino acid to reduce the non-specific 12:07 binding between the cas9 and the dna 12:14 the sp cas9 gene is too big to be inserted with a single guide rna inside an adeno 12:20 associated virus and thus researchers have identified cas9 from other bacteria 12:27 that are smaller for example this staphylococcus probably is cast 9 it is smaller than 12:33 the sp 9 but it has a different path which is more restrictive 12:41 another type of crispr cas9 enzyme has been identified it's called cpf1 it produces sticky 12:48 cancers it's another one cut of the dna 12:54 there is just a whole series of various castings obtained from various bacteria 13:00 that require different types the 13:06 crisprcas9 technology may be used not only to reduce cuts in the dna but 13:11 it may also be used to induce the expression of a gene by fusing cast 9 with bp 13:16 64 for example or iron with crab to repress the expression of a gene 13:26 in my research group we are using the crispr cas9 technology to develop therapies for various disease 13:33 much ataxia design muscular dystrophy and alzheimer's disease i will present that now 13:41 we have initially used the crispr cas9 technology to develop a treatment for freshwater 13:47 taxi it is an adidas disease due to the presence of a long 13:53 tri-nucleotide with bgaa in intron one of the fertilizer gene this 14:00 long repeat reduce the expression of the fataxin gene leading to 14:07 neurological problems and cardiac problems we have just used the crispr cas9 14:13 technology and generated single guide rna able to cut in intron 1 before 14:21 and after the trinucleotide repeat leading to its removal and increasing the expression of ataxia 14:31 indeed the removal of the tri-nucleotide repeat in cells of the yg8 14:38 sr mouse model of frederic ataxia doubled the expression of fataxin 14:44 compared to the untreated cells and raise the expression of protection to 14:50 almost the normal level the sp cas9 gene is too big to be 14:56 delivered with two single gut rna by a single aav 15:01 we are just currently using the smaller cgcas9 gene to remove the trinucleotide in intron 15:09 one of the fataxin gene using this smaller cg cas9 nuclease 15:18 we are also able to remove the trinucleotide repeat in intron 1 of the fratexin gene 15:27 we are also using the crispr cas9 technology to develop a treatment for duchenne 15:33 muscular dystrophy duchenne muscular dystrophy is due to 15:38 mutation in a gene coding for the dystrophin protein it is a large gene containing 79 exons 15:47 some of these exons do not contain a multiple of three nucleotides and thus seventy 15:53 percent of duchenne muslim dystrophy cases are due to deletion of one or several exon and the total 16:00 number of coding nucleotides which are deleted is not a multiple 16:05 of three for example in this presentation we have 16:12 a deletion of exon 50 that does not contain a multiple of three nucleotides 16:18 this lead to a frame shift and there will be a stop codon in exon 51. 16:24 thus the beginning of this the dystrophin protein is expressed but not the end of the dystrophin 16:30 protein this situation may be corrected by inducing the skipping that is the 16:36 removal of exon 51 in the messenger rna 16:41 this is done using antisense oligonucleotide this exon 51 may also be deleted 16:50 by inducing cuts in intron 50 and in front 51 to completely remove 16:56 that exon this results in the expression of the beginning of 17:02 the dystrophin protein and of the end of the dystrophin protein but there is however 17:08 a small part of the protein which is missing in the center of the protein the removal 17:15 of one or several complete exam may thus restore the reading frame and 17:21 convert a duchene patient into a becker patient however some backer patients have severe 17:29 symptoms and are bound to a wheelchair at the age of 11. 17:34 therefore the improvement of some duchenne muscular dystrophy may not be significant 17:42 this is because the dystrophin protein has a complex structure indeed in its central part 17:49 the dystrophin protein contains 24 spectrum-like repeats 17:56 each spectrum ligatude is made of three alpha helix helix a linux b 18:03 and exc note that nx a is starting on the left side and elix c 18:09 is ending on the right side and normally there is a succession of abc abc abc 18:19 the main problem of deleting complete exam to restore the expression of dystrophin protein is 18:26 that the beginning and the end of the spectrum-like repeat indicated in this scheme by the black 18:33 arrow do not correspond with the beginning and the end of exiles and thus when removing complete absorbs 18:41 the resulting of the spectrum-like repeat structure may not be normal 18:49 vector muscularis computation have a deletion of one or several exon but the total 18:55 number of coding nucleotides which are deleted is a multiple of three nucleotides and thus there is 19:02 no frame shift this is the case for example for becker patient having a deletion of example 45 19:09 to 47 or having a deletion of 45 to 49 in both case there's no frame shift the 19:16 beginning and the end of the dystrophin protein are expressed however at the junction site between the 19:24 remaining codons there is an abnormal structure of the dystrophin protein and 19:31 due to this abnormal structure of the dystrophin protein these vector patients are bound to a 19:36 wheelchair at an early age 19:43 thus in my research group instead of trying to restore the normal reading frame 19:48 by deleting complete exons we have instead aim at producing an evaline exam 5054 19:56 which not only restore the normal reading frame but calls for a dystopian protein with a 20:03 normal structure we have done our initial experiments using the myoblast of a 20:08 duchenne patient having a deletion of exam 51 52 and 53. 20:14 thus because the number of coding nucleotides was not a multiple of three there was a stop codon 20:22 in exam 54 due to the frame shift we have just used a crisper cas9 20:28 technology to induce a cut in exon 50 and a cut in exon 54 to create the e-braid exam 50-54 20:39 to produce this immigration exam we initially identify what are the possible sp cas9 20:46 pam or what other site where we can cut in exon 50 and in exon 54 we identify 20:54 10 time sites in exam 50 and 14 different pan size in exon 53. 21:02 this table predicts what are the results of inducing cuts in exam 50 21:08 and in exam 54. when we have a blue square it means that the 21:16 cut has been produced in exon 50 right after the three nucleotides and 21:22 calls for an amino acid and the cut has been produced in exon 54 21:29 just before the three nucleotide that codes for an amino acid thus at the junction point we have amino 21:37 acids that are one escorted by example 54 followed immediately by an amino acid 21:44 coded by example there's no friendship and there is no new amino acid produced 21:51 however when the square is clean that means that we have at the junction 21:58 site a new amino acid which is produced because we are cut in exon 50 22:04 right after one of the nucleotides of the codon and the two nucleotides that will 22:09 complete these codons are from exotic before we have cut 22:14 right after one nucleotide of the codon in exam 54 and thus first two nucleotides left 22:22 to create a new codon at the junction site there's just a new amino acid at the 22:28 junction site but there is no frame shift and all the other amino acids 22:33 are the correct ones it is also possible with this square in red to have a new 22:40 codon produced at the junction side but this new codon is a stop codon this 22:46 is not real we want to do all the squares in wide because 22:52 there is a frame shift we're cutting after one nucleotide of the codon in exon 15 22:57 and we're cutting before the last nucleotide of the cooldown in exam 54 and thus add the junction 23:06 side there is a new codon which is done because there is a frame shift 23:11 and all the amino acids that follows are not the correct one 23:18 we have dust testers there your sphere of guide irony one guide irony cutting in zone 50 and 23:25 the other guide ironing cutting in exon 54 this produced an ibrit exam 1554 23:32 that had the predicted size of this hybrid exam note that when we 23:39 are doing cuts in exam 50 and in exon 54 in a normal dystrophin g 23:45 we are deleting 160 000 base pair and despite that the junction between 23:52 exon 50 and 54 is of the size predicted we have sequence 24:01 this elite exam that was produced by cutting in exon 50 and in exon 54. not only 24:08 were the eblid exam exactly the size as predicted 24:14 but the sequence were also exactly as predicted for example we had a junction site the 24:21 fusions of exon 50 and example 54 producing exactly the predicted amino 24:29 acid we also obtained at the junction site a new codon coding for the predicted 24:36 amino acid or coding for a stop codon and sometime as predicted by the white 24:43 square we had a frame shift and when there was a frame shift there were stub cooldowns that were met 24:51 in the resulting exams 24:58 as mentioned before the dystrophin protein contained in its central part 24 spectrum like 25:05 repeat each one being made of three alpha helix a b and c we have just used a guide rna to 25:13 induce a cut in exon 50 in a sequence coding for lxc 25:19 and another guide rna inducing a cut in exon 54 also in a sequence coding for helix c 25:26 the resulting evidence exon 5054 calls for an ibrahim 25:33 alex c where the beginning of nxt is coded by exon 50 and the end 25:40 of lxc is coded by exon 54. this is a structure that has been 25:45 computer predicted using the sequence of the resulting protein 25:54 when the bioglass of this duchenne patient having a deletion of exam 51 to 53 26:00 are fused together to form some small muscle fibers and culture called myotubes in fact these muscle 26:07 fibers do not express dystrophin because this is a shame patient however the myoblast of a 26:13 healthy human when they fuse together they form a small biotube and they do express this 26:19 topic as can be seen here in this western blood when we use the bioblast of the duchenne 26:25 patient and we induce the formation of the ignition exam 50-54 we have expression 26:32 of the dystrophin protein in culture and as you can see this dystrophin protein 26:38 has a lower molecular weight than the normal dystrophin because there is deletion of exon 51 to 53 26:46 and an additional deletion of part of exon 50 part of the exam 54 thus there is a part 26:53 of the dystrophin protein which is absent and this is why the protein is smaller 26:59 but it is nevertheless expressed for our initial in vivo experiments 27:06 we have used the hdmd mouse model this is a transgenic mouse that 27:12 expressed the human dystrophin gene all introns in all exons 27:18 we have electroporated in the muscle of this mouse plasmid coating for the sp cas9 27:26 fuels with the fluorescent green protein and two single guide rnas this 27:33 a month later when we took the muscle there was expression of the green 27:38 fluorescent protein confirming that the plasmid 27:43 had been correctly electroporated in the muscle fibers leading to the expression of the 27:49 fluorescent green protein and probably also leading to the expression of the sp cast iron 27:55 and after two glide ironing we then extracted the dna from these 28:02 muscle and we first confirmed using a test called the surveyor enzyme 28:08 that indeed they were constantly being produced in exam 50 and 54. we then 28:15 use pcr to amplify the ebrade exon 5054 28:21 note that in the muscles that are not treated with the crispr cas9 technology 28:26 there is no amplification of example 5054 because there are 160 000 base pair 28:33 between these two exons and therefore the two primers for the pcr are too much 28:40 separated from one another 28:50 we then sequence this exon 5054 that was produced in vivo 28:58 in the mouse muscle expressing the human dystrophin gene and as predicted we had the sequence of 29:05 exon 50 and a new codon at the junction site 29:10 followed by all the correct codon coding for the normal amino acid 29:16 in exon 54. this is exactly as predicted in vivo 29:24 however for a delivery of the cas9 gene in vivo to several muscle 29:32 we need to use an adeno associated virus as mentioned before the sp cas9 29:38 g is too big to be delivered with two guide irony by a single aav and we therefore use for 29:46 our next experiments the cast 9 of the staphylococcus moleus 29:51 which is a smaller cas9 which permit delivery with two guide rna by a single avp 29:58 in this case we have used a guide rna that conducts cuts 30:05 in exon 47 in a sequence coding for elix b and in exon 58 30:12 in a sequence again coding for elix b this resulted in the formation of an 30:18 ebola exome 4758 coding for an ibrigid 30:24 hxb which has a normal structure the beginning of the lxb being coded 30:30 by exon 47 and the end of that evil 30:36 be encoded by exon 58 30:42 we then use for our indivo experiment a new mouse model called hdmp delta 30:50 ii it's the same mouse model as previously that contains 30:55 the dystrophin gene with all the exons and all the intron except that 31:01 this mouse has a deletion of exam 52 and thus there is no expression of the human dystrophing gene 31:09 we have thus used the crispr cas9 technology to induce a cut in exon 47 31:15 and a cut in exam 58 producing the eberlini exam 4758 31:21 resulting in the production of a dystopian protein containing an ibrite example 31:28 in lxp we have injected to these hdmd delta 52 31:38 mouse aev's coding for sc cast 9 and 2 single guide rna 31:43 a month later we observe in the muscle the expression of the human dystrophin gene including 31:51 in the heart we are just proposing a treatment of 31:56 duchenne muscular dystrophy which would be the systemic delivery by an adeno associated virus 32:03 of the class 9g and of two guide rna to form any bread exam in contrast 32:10 with exam skipping which is a treatment done at the level of the message rna the treatment that we 32:16 propose at the level of the dna would be permanent 32:22 the crispr cas9 technology is evolving rapidly and new technologies derived from the 32:28 crispr cas9 permits now the modification of a single nucleotide 32:36 more than 32 000 single nucleotide modifications are responsible for 32:43 hearing steady disease and thus the capacity to correct a single nucleotide 32:49 would provide treatment for most of these healing steady disease 32:55 the first treatment which permit the modification of a single nucleotide was developed by command in 2017 33:04 which used a cas9 knee case that is the modified class cas9 which is able to cut 33:11 only one strand of dna and this cast 90 case is fused 33:16 with acetidine deaminase this technology impermanently clinically 33:23 modify city bean into a uvd which is replaced 33:28 by a timing of dna repair 33:34 the main limitation of that technique is the chemical modification of the ctd will 33:40 occur in a narrow window located at 12 to 16 base pair from the 33:47 ngg pan we have initially used base editing 33:55 technology to develop a treatment for alzheimer's disease 34:04 the alzheimer's disease is produced by an abnormal metabolism of the ammunition 34:12 protein normally this protein is cut by the alpha secretaries 34:18 followed by a cut by the gamma symmetries this produce peptides and protein 34:24 fragments that are degraded without causing any problem however this protein may also be cut by 34:31 the beta sequence followed by a cut by the gamma secretaries and this produced 34:37 short 40 42 amino acid long beta middling peptides that 34:44 aggregates to one another forming amino acids that interfere with synaptic 34:50 transmission leading to neuron death and the memory problems 34:58 this scheme illustrates the amino acid sequence of the transmembrane part of the 35:04 anaerobic repressure protein we can see the position of the beta alpha 35:09 and gamma separates outside all the amino acids the star above their 35:15 name are amino acids which are quantified leading to family of form version of disease please 35:24 note the position with the red arrow this is the alanine in version 673 35:30 when this adenine is changed by valley this leads to severe early onset alzheimer's disease and you 35:38 are azamara at the age of 40. however when this adenine is 35:47 105 years old as shown by johnson nepal in nature 2012. 35:57 the presence of the a673t mutation also known as the icelandic 36:03 mutation reduce the secretion of a beta 40 in our beta 42 peptide 36:10 for the wild-type eppg and for appg containing the london mutation 36:20 our experiments have shown that the presence of the a673t mutation 36:27 reduce the secretion of a beta 40 and a beta 42 peptide by the epp 36:35 genes not only for the wild-type gene but also for app genes containing several familial 36:42 alzheimer's disease mutations 36:49 as mentioned before the crispr cas9 base editing technology 36:54 permit to modify the city into a timing 37:02 we just have used the base editing technology to target the cytoplane 37:10 in the antecedent strand of the alanine codon transforming that cytodine into a timing 37:16 and just changing the alanine codon into a triangle 37:25 the main problem with this approach is that although we want to modify the cytodine 37:32 into the end descent strand of the adenine codon there are other acidity nucleotides 37:40 nearby that are also affected by the base editing approach 37:48 we have constructed 14 different base editing enzymes to be able to modify 37:55 more specifically in the antisense cooldown of anatomy 38:04 modifying the city and the antisense codon of led we have been able to introduce the h673t 38:12 mutation in up to 17 of the avpg however 38:19 other city also located in the antecedent strat were also modified by this approach 38:29 a new fantastic technology called private has recently been developed by enceladus 38:36 with permits in principle to replace any nucleotide by any 38:45 the other editing technology use a cas9 decays fused with a reverse 38:52 transcriptase it also requires a prime editing guide rna known as a pig remedy 39:05 the peg rna is essentially prolonged single guide rna because as a single 39:11 guide rna it contains a spacer sequence which react with 20 nucleotide 39:18 in the target dna it also contains the constant scaffold of the single 39:24 guide rna which is in red and then at its uh five prime end there is a 39:31 prolongation with the primer bending side which is a sequence of 10 to 39:37 17 nucleotide reacting with the upper strand of dna 39:42 this is followed by the reverse transcriptase template again which is 10 to 17 nucleotides in 39:50 length and which will contain some modified nucleotide in red in this case 39:57 indicating which nucleotide has to be modified by the reverse transcriptase 40:06 a plasmid designed by anzalone at all 40:12 is available at edgy to construct new peg rna 40:22 this classmate contains a red fluorescent protein gene which may be removed by bsa1 cuts which 40:28 produce a backbone of the protein and then the other components are the spacer primer binding side the reverse 40:35 transcriptase template and the pig rna scalpel all of these sequence are single 40:40 stranded oligonucleotide that may be purchased from idt these four parts are then assembled 40:47 together to produce a new pegaron 40:53 to use the prime editing technology we first have to identify photo spacer adjacent tab 41:01 which is njj for the cast nine of streptococcus pyrogene 41:08 this will permit to the cas9 to bind to the dna then the spacer sequence of the peg rna 41:15 will bind to a 20 nucleotide sequence of dna in this case the lower strand 41:20 and the formation of the complex between the peg rna class 9 decays and the dna 41:28 will induce the nick at exactly 3 nucleotides from the pan and the upper strand of dna this will 41:36 release the upper strand of dna to be able to interact with the primer binding side of 41:43 the peg rna and then the reverse transcriptase template 41:49 which contained few nucleotides to be related will be available for the 41:55 reverse transcriptase to synthesize a new dna upper strand 42:04 many duchenne muscular dystrophy patients have a stop codon dmdg since we did not have access 42:13 to cells of patient containing such pointation we decided to introduce these stop 42:20 codons using the prime editing technology so each case we had to identify an ngg 42:29 identify the peg rna protospacer sequence and then modify the reverse 42:36 transcriptase template so as to enter to modify a 42:41 codon for an amino acid into a stockholder in this case we have changed 42:51 to introduce the tga stuff 42:57 we have successfully used that approach to introduce stun codon exam 9 43:03 20 35 42 55 and 61. 43:12 have just designed various pig rna targeting dmd exon 35 as you can see 43:19 we have varied the reverse transcriptase template in blue from 10 to 16 nucleotide 43:27 and we have also varied primary binding site in green from 10 to 15 nucleotides 43:35 and at the desired mutation site we have introduced a t to 43:42 introduce that mutation 43:48 our initial experiments were done in hgk 293 cells 43:55 we tried to reproduce the mutation of mx1g and to target 44:03 dmd exon 35. so essentially these cells were transfected 44:08 with plasmid coding for the cas9 djs fuse with the reverse transmitted days 44:14 and a paid rna targeting either exon 35 of the dmd gene 44:20 or the emh one gene the dna was extracted three days later 44:27 and the targeted sequence was pcr amplified and sequenced using center method 44:34 the sequence were analyzed using the edit r online program essentially we observe 44:41 a 32 correction mutation of the air x1 gene as 44:48 done by heads alone at all however for the exon 35 mutation 44:55 we had a 2 background in the sequence of the control negative 45:02 control and with the different peg rna we have mutation ranging from four to eight 45:10 percent so this was not as great as for the e mx one g 45:20 we have just tried different methods to try to increase the percentage of genome editing 45:26 of exon 35. the first method that we have tried is to repeat the treatment three times 45:33 essentially the cells were transfected with plasmid at day 0 6 12. 45:40 and dna was extracted three days and six days after each treatment 45:48 we have amplified the pcr exon 35 at each of the extraction date 45:55 and sequence it by sender and analyzer sequence as you can see the percentage of genome 46:02 editing increased from day 3 to day 18 with the repeated treatment 46:08 and this was the case for all three peg rna that were targeting 46:18 we have then tested a second method to try to increase the percentage of mutation in exon 35. 46:26 it is to induce a second dig in the target gene this is the pe3 method 46:33 essentially we have identified two pam sequence which permitted to kite rna to induce 46:40 a second nick and either 57 nucleotides from the original 46:47 big heavenly name or at 24 cleotide from the pagan herd 46:57 when we induced a second leg at 24 nuclear time or 47:04 at 57 nucleotides induced by the peg allergy there was a 47:10 significant increase in the addition of the target gene for pig iron in the 35 47:16 4 and pig rna 256 but not for bigger and e35 you still have to 47:23 understand why 47:30 a third method to improve the percentage of mutation in the target gene is to mutate 47:37 the pan used by the peg harmony 47:44 we just designed pig rna that were not only able to introduce a stop codon 47:50 mutation but we're also able to mutate simultaneously 47:56 the pan and the use 48:02 mutation of the pan used by the peg rna improve the percentage of mutation in 48:08 the stop codon for two of the three peg rna that we 48:14 have tested 48:20 combining the two method that is inducing a second nick in the target g 48:26 and mutating the pan used by the peg rna further increase the mutation of dmd 48:34 exon 35 to 39 with all three peg that we have 48:46 tested we are currently starting new project to correct mutation 48:53 responsible for other heading steady disease we are working on cystic fibrosis due to 48:59 mutation in the cftr chloride channel on congenital muscular 49:04 dystrophy due to mutation in the rheanodine receptor and on 49:10 ataxia 8 due to mutation in the nkx6 type 2 gene 49:20 for each period steady disease due to a point mutation for example here with attacks at 49:27 type 8 it is possible to correct in principle the mutated gene using the prime editing 49:33 technology in this case here we can identify that the mutation is an adenosine change for timing 49:41 and thus we can identify a pam njg for the sp cast iron which is close to the mutated nucleotide 49:49 we can then design a plane rna that will introduce the desired mutation in this 49:55 case we are introducing two mutations one to correct the mutation to reverse the timodine 50:02 into an adenosine and the second midpoint mutation 50:07 is to modify the path so that following the correction the cad9 enzyme can no longer bind to 50:15 the dna crispr cas9 derived technologies may not 50:24 be used to treat many individual diseases the main problem remains the inhibitor 50:29 delivery of the editing agents 50:35 thank you for your attention 50:43 okay thank you professor jax uh i'll take the turn now to make a general comment for those who 50:52 the who doesn't speak uh those who don't speak english okay thank you for your presentation 50:58 thank you of course [Music]