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4:27 good evening from sword jacksonville uh it's a great honor to hear your 4:32 words about crispr this birth i i could say crispr 4:39 is an intriguing chapter of medicine it's a an intriguing chapter of life 4:47 and i i i'm sure that this admit you will be successful and 4:55 i'd like to thank you and dr rabio lanot for accepting the invitation 5:04 now i'm going to uh to share my screen to begin my my talk 5:36 foreign 6:23 [Music] 6:32 foreign 6:39 [Music] 7:09 [Music] 7:42 um 8:18 [Music] 9:03 [Music] 9:17 [Music] foreign 9:53 [Music] 10:29 [Music] 11:40 uh 12:06 [Music] 12:36 is 13:32 a 15:34 [Music] 16:04 [Laughter] 17:02 foreign 17:59 is 18:08 [Music] 19:18 [Music] 20:18 um 20:39 [Music] 20:46 start your speech welcome to the symposium thank you for the invitation 20:54 let me start my presentation 21:00 research work using the crispr cast 9 technology 21:09 today's presentation will be divided into part i will first present the use of the 21:15 crispr cas9 technology to treat iron steady disease and my graduate 21:20 student gabrielle lamont will present all the crispr cas9 derived 21:25 technology may be used to detect viral infection 21:32 the word crispr stands for clustered regulatory interspace short penetrate repeats 21:41 whisper cas9 technology is derived from research on bacteria it was initially discovered in 2005 21:49 that the crispr sequence contained viral sequence and yet there was a 21:54 hypothesis that crispr cas9 is the bacterial immune system 22:00 it's only in 2013 that it was discovered that the crispr cas9 22:06 may be used to induce specific modification of the human genome 22:11 and this started a big explosion of artificial 22:17 indeed the crispr casting system is used by bacteria to kill bacteriophage bacteriophage are 22:24 extremely abundant they're ten times more abundant in bacteria and there is a war that is ongoing between 22:32 bacteria and bacteriophage since millions of years 22:39 it was initially discovered that they are in the genome area constant repeated sequence 22:47 researchers were wondering what these repeated sequences were for and then they discovered that the 22:54 sequence between a constant repeat were sequenced that were up from the genome of various 23:01 bacteriophage in fact what normally happens is when a 23:07 bacteriophage infected bacteria normally the bacterial ph wind bacteria is dead 23:13 but sometimes you have a defective virus that will infect without killing the bacteria but they will then 23:19 be able to acquire some part of the sequence of the virus 23:24 and it will store that into its genome and it will be ready when it needs such bacteriophage again 23:31 in the future from the dna in the crispr area bacteria 23:37 will first express the pre-cr powder and then there will be a complex that will be made between 23:43 tracker or crna and the cast iron protein 23:49 this striker rna crna cas9 protein complex will then bind with the bacteria of hdna 23:57 and induce a cut of the bacteriophage dna to essentially kill the virus 24:07 to bind to the dna the cas9 protein requires the presence of the total spacer 24:12 adjacent motif which is simply njj for these streptococcus pyrogene 24:19 path then there is a crrna 24:24 which contain a variable sequence of 20 nucleotides that will be 24:30 complementary to the bacteriophage sequence and then there is also the tracker rna which is a constant 24:36 rna sequence that forms a complex with the crm when the three parts are together cr rna 24:44 tracker or a cast iron protein a cut will be induced at exactly three nuclear 24:50 tight from the plant of course since this is a war there are 24:56 contour measure that are taken by the phage they will mutate their genome so that they become 25:02 resistant not recognized by the cr rna and will survive 25:08 bacterial defense the crispr cas9 big bang started in 2012 25:17 when gen x and colleagues realized that the crisper cast 9 technology can permit to 25:23 cut the genome of planets and animals they also fuse their cr rna and the tracker rna 25:31 together to form a single guide rna this was done essentially because 25:38 the single guide rna is something that can be patented whereas the crn in the tracker 25:44 being natural they cannot be patented 25:50 during the following year there was an explosion of article confirming that the crisper casino 25:56 technology may be used to cut the genome of bacteria human cells carbon animal mouse drugs 26:02 zebrafish plant flies nematodes monkeys 26:10 many of these articles were published in prestigious journals such as nature 26:15 science nature biotechnology molecular cell cell molecular cell 26:23 there was a rapid explosion of article and as you can see as of november 26 2020 26:31 21 420 articles have been published where the word crispr is mentioned 26:40 the main reason of this rapid explosion of article is that it costs much less to derive 26:47 a sequence that will be able to cut a specific genome sequence compared to other 26:53 pre-existing technology such as zinc finger protein and tailors 27:00 as mentioned before the binding of the cas9 protein to the dna 27:06 require the presence of a photo spacer adjacent onsite pam which is njj for the cas9 derived 27:12 from streptococcus biology there is then the binding also of the 27:18 single guide rna which will recognize the sequence of 20 nucleotides 27:24 and when the complex is made between the cas9 protein the single guide rna and the dna there 27:31 is a cut that will be made at exactly three nucleotides from the pan 27:39 once inside the nucleus the resulting complex will lock onto a short sequence known as the pan 27:46 the cast 9 will unzip the dna and match it to its target rna 27:53 if the match is complete the cas9 will use two tiny molecular scissors to cut the 28:00 dna when this happens the cell tries to repair the cut 28:06 but the repair process is error-prone leading to mutations that can disable the gene allowing researchers to understand its 28:14 function these mutations are random but sometimes researchers need to be more precise 28:20 for example by replacing a mutant gene with a healthy copy this can be done by adding another piece 28:27 of dna that carries the desired sequence once the crispr system has made a cut 28:33 this dna template can pair up with the cut ends recombining and replacing the original sequence with the new version 28:46 one of the reasons of the rapid explosion is that the crystal classic technology also used all what was developed by gene 28:53 that have been the 30 years that preceded it we have methods to delivering the dna 28:59 using aav and then associated virus cationic polymers liposome micro 29:05 injection electro operation all of those techniques are used in the context of crispr 29:12 when a double strand break is produced in the dna most of the breaks will be repaired by 29:18 non-homologous adjoining that will lead to micro deletion or micro insertion this is frequently 29:26 used to knock out the expression of a gene by changing the reading frame 29:32 and it can also be repaired by homology directed repair which require the presence of the donor 29:37 dna that contains sequence of homology with what is receding what is following 29:43 the cut and then between the two homology sequence there is a sequence in blue 29:49 which may be a single nucleotide or a whole gene that will be inserted at the side of the 29:55 double strand break when we use the crispr class 9 30:00 technology we intend to produce a double strand break at a precise site in the human genome 30:07 however the cast 9 may sometimes induce cut at other sites in the genome which are 30:13 called buff target mutation computer software may predict these off-target mutation by using 30:20 the whole human genome sequence however these softwares are not perfect 30:25 and they will sometimes print it off target mutation at site where there is no cut 30:31 and sometimes there will be a cut at the side which is not predicted by the computer 30:39 there are or however some experimental techniques such as a guidestack method which permit 30:46 to experimentally identify the sites of target mutation this guidesec 30:52 technique used the introduction of a short 34 base pair oligonucleotide 30:59 at the sides of the cuts and later by pcr the site of insertion may be identified 31:05 by sequencing the presence of these off-target 31:10 mutation is the main problem that delays the use 31:15 of crispr cat9 technology for direct vehicle corrections 31:23 one of the method to reduce of target mutation is simply to reduce by two or three the 31:29 number of of the single guide rna which is binding with the target dna 31:39 another method is to use a mutated cas9 nuclease that cut only one strand of dna is then 31:46 called the knee case there is variation of cas9 where the decays cut only the 31:53 lower strand of dna and other mutation of class 9 which permit to cut only the upper 32:00 strand of dna it is thus possible to induce more 32:05 precise double strand break in the dna by using the cas9 nikkei 32:12 and two single guide rna each detecting a sequence of 20 nucleotides close to 32:17 one another [Music] it is also possible to use a 32:23 non-functional cas9 cas9 nuclease fuse with the one 32:29 nucleates then two guide rna are again needed to detect sequence which are close to 32:36 another and the frog one nuclease need to form a dimer in order to be able 32:41 to cut the dna finally some researchers have mutated 32:49 the gene coding from cast 9. they have modified some sequence coding for amino 32:57 acid to reduce the non-specific binding between the cas9 and the dna 33:06 the sp cas9 gene is too big to be inserted with a single guide rna inside an adeno 33:13 associated virus and thus researchers have identified cas9 from 33:18 other bacteria that are smaller for example the staphylococcus it is smaller than the sp 33:27 9 but it has a different path which is more restrictive 33:33 another type of crispr cas9 enzyme has been identified it's called cpf1 33:39 it produces sticky cancers it's another blood cut of the dna 33:46 there is just a whole series of various cas9 containment values with kylia 33:52 that require different tabs 33:58 the crispr cas9 technology may be used not only to reduce cuts in the dna but it may also be used 34:04 to induce the expression of a gene by fusing cas9 with bp 64 for example 34:10 or choosing cast 9 with crab to redress the expression of a gene 34:18 in my research group we are using the crispr cas9 technology to develop therapies for various disease 34:25 transverse ataxia duchenne muscular dystrophy and alzheimer's disease i will present that now 34:33 we have initially used the crispr cas9 technology to develop a treatment for fredrich 34:39 ataxia it is an elixir disease due to the presence 34:44 of a long try nucleotide with b gaa in intron one of the fataxin gene 34:52 this long repeat reduce the expression of the fataxin gene leading to 35:00 neurological problems and cardiac problems we have just used the crispr cas9 35:05 technology and generated single guide rna able to cut in intron 1 before 35:14 and after the trinucleotide repeat leading to its removal and increasing the expression of ataxis 35:24 indeed the removal of the trinocleotide repeat in cells of the yga 35:31 sr mouse model of frederic ataxia doubled the expression of frataxin 35:36 compared to the untreated cells and raised the expression of protection to almost 35:43 the normal level the sp cas9 gene is too big to be 35:49 delivered with two single guide rna by a single aav we are just currently using the smaller 35:56 cg cas9 gene to remove the trinucleotide repeat in intron 1 36:02 of the fataxin gene using this smaller cg cas9 nuclease 36:10 we are also able to remove the trinucleotide repeat in intron one of the frataxin gene 36:20 we are also using the crispr cas9 technology to develop a treatment for duchenne 36:25 muscular dystrophy duchenne muscular dystrophy is due to 36:31 mutation in a gene coding for the dystrophin protein it is a large gene containing 79 exons 36:39 some of these exons do not contain a multiple of three nucleotides and thus 70 percent 36:46 of duchenne muscular dystrophy cases are due to deletion of one or several 36:51 exams and the total number of coding nucleotides which are deleted 36:56 is not a multiple of three 37:01 for example in this presentation we have a deletion of exon 50 that does not 37:07 contain a multiple of three nucleotides this lead to a frame shift 37:12 and there will be a stop codon in exon 51. thus the beginning of this the 37:18 dystrophin protein is expressed but not the end of the dystrophin protein 37:24 this situation may be corrected by inducing the skipping that is a removal of exon 51 37:32 in the messenger rna this is done using antisense polygon nucleotide 37:38 this exam 51 may also be deleted by inducing cuts in intron 50 and 37:46 in in front 51 to completely remove that exon this results 37:52 in the expression of the beginning of the dystrophin protein and of the end of the dystrophin protein 37:59 but there is however a small part of the protein which is missing in the center 38:04 of the protein the removal of one or several complete exam 38:11 may thus restore the reading frame and convert a duchene patient 38:16 into a becker patient however some backer patients have severe 38:21 symptoms and are bound to a wheelchair at the age of 11. 38:26 therefore the improvement of some duchenne muscular dystrophy may not be significant 38:35 this is because the dystrophin protein has a complex structure indeed in its central part the distro 38:42 fin protein contains 24 spectrum-like repeats 38:49 each spectrum like repeat is made of three alpha helix helix a lxb 38:55 and lxc note that lxa is starting on the left side and elixi 39:02 is ending on the right side and normally there is a succession of abc abc abc 39:11 the main problem of deleting complete exon to restore the expression of dystrophin protein is 39:19 that the beginning and the end of the spectrine like repeat indicated in this scheme by the black 39:26 arrow do not correspond with the beginning and the end of exiles and thus when removing complete exhaust 39:33 the resulting of the spectrum-like repeat structure may not be normal 39:41 vector muscular dystrophy patient have a deletion of one or several exams but the total 39:48 number of coding nucleotides which are deleted is a multiple of three nucleotides and 39:54 thus there is no frame shift this is the case for example for becker patient having a deletion of exam 40:00 45 to 47 or having a deletion of 45 to 49 40:06 in both case there's no frame shift the beginning and the end of the dystrophin protein 40:12 are expressed however at the junction site between the remaining codons there is an abnormal 40:20 structure of the dystrophin protein and due to this abnormal structure of the dystrophin protein 40:26 these vector patients are bound to a wheelchair at an early age 40:35 dusk in my research group instead of trying to restore the normal reading frame 40:41 by deleting complete exons we have instead aim at producing enabling exam 40:47 5054 which not only restored a normal reading frame but codes for a dystopian protein with a 40:55 normal structure we have done our initial experiments using the myoblast of a 41:01 duchenne patient having a deletion of exam 51 52 and 53. thus because 41:08 the number of coding nucleotides was not a multiple of three there was a step codon in exon 54 41:16 due to the frame shift we have just used the crisper cast 9 technology to induce a cut in exon 50 and a cut in 41:24 exon 54 to create the ebrate exon 5054 41:31 to produce this implicated example we initially identify what are the possible sp cas9 41:39 pan or what are the site where we can cut in exon 50 and in exon 54 41:45 we identify 10 times sites in exon 50 and 14 different pan size in exon 53 41:54 this table predicts what are the results of inducing cuts in exam 50 42:01 and in exam 54. when we have a blue square it means that the 42:09 cut has been produced in exon 50 right after the three nucleotide and 42:15 calls for an amino acid and the cut has been produced in exon 54 42:22 just before the three nucleotide that codes for an amino acid thus at the junction point we have amino 42:29 acids that are one escorted by exon 54 followed immediately by an amino acid 42:36 coded by example there's no frame shift and there is no new amino acid 42:43 however when the square is clean that means that we have at the junction 42:50 site a new amino acid which is produced because we are caught in exon 50 42:56 right after one of the nucleotides of a codon and the two nucleotides that will 43:01 complete these codons are from example before we have cut 43:06 right after one nucleotide of the codon in exon 54 and thus first two 43:12 nucleotides left to create a new codon at the junction site there is thus a new 43:19 amino acid at the junction site but there is no frame shift and all the other amino acids are the 43:26 correct ones it is also possible in this square in 43:31 red to have a new codon produced at the junction site 43:36 but this new codon is a stop codon this is not real we want to do all the squares in wide because 43:44 there is a frame shift we're cutting after one nucleotide of a codon in exon 15 43:50 and we're cutting before the last nucleotide of a cooldown in exon 54 and thus add the junction 43:58 side there is a new codon which is done because there is a frame shift and all 44:04 the amino acids that follows are not the correct one 44:10 we have dust testers various pair of guide irony one guide irony cutting in at zone 50 44:17 and the other guide rna cutting in exon 54 just produced an ibrit exam 1554 44:24 that had the predicted size of this hybrid exam note that when we 44:31 are doing cuts in exam 50 and in exam 54 in a normal dystrophin g we are deleting 44:39 160 000 base pair and despite that the junction between 44:45 exon 50 and 54 is of the size predicted 44:52 we have sequenced this hybrid exam that was produced by cutting in exon 50 and 44:58 in exon 54. not only were the eblid exam exactly the size as 45:05 predicted but the sequence were also exactly as predictive 45:10 for example we had a junction site the fusions of exon 50 in exam 54 producing 45:19 exactly the predicted amino acid we also obtain at the junction site a new codon 45:27 coding for the predicted amino acid or coding for a stop codon and 45:33 sometime as predicted by the white square we had a frame shift and when there was 45:39 a frame shift there were stop codons that were meant in the resulting liberal exiles 45:50 as mentioned before the dystrophin protein contained in its central part 45:56 24 spectrum like repeat each one being made of three alpha helix a b and c 46:03 we have just used a guide rna to induce a cut in exon 50 in a sequence coding 46:10 for lxc and another guide rna inducing a cut in exon 54 46:15 also in a sequence coding for helix c the resulting evident exon 5054 46:22 calls for an ibrim elixi where the beginning of nxc 46:29 is coded by exon 50 and the end of elixi is coded by exon 54. 46:36 this is a structure that has been computer predicted using the sequence of the resulting 46:42 protein when the bioglass of this duchenne 46:48 patient having a deletion of exon 51-53 are fused together to form some small 46:55 muscle fibers and culture called myotubes in fact these muscle fibers do not express dystrophin because 47:02 this is a duchenne patient however the myoblast of a healthy human 47:07 when they fuse together they form a small biotube and they do express this topic as can be seen here in this western 47:14 blood when we use the bioblast of the duchenne patient and we induce the formation of the 47:21 ignitive zone 5054 we have expression of the dystopian protein in culture 47:27 and as you can see this dystrophin protein has a lower molecular weight than the normal 47:33 dystrophin because there is deletion of exon 51 to 53 47:39 and an additional deletion of part of exon 50 and part of the exam 54. thus there is a 47:45 part of the dystrophin protein which is absent this is why the protein is 47:51 smaller but it is nevertheless expressed 47:56 for our initial individual experiments we have used the html mouse model 48:02 this is a transgenic mouse that express the human dystrophin gene 48:08 all introns and all exons we have electroporated in the muscle of 48:14 this mouse plasmid coating for the sp cas9 fuels with the fluorescent green protein 48:21 and two single guide rnas this a month later when we took the muscle 48:28 there was expression of the green fluorescent protein confirming that the plasmid has been 48:36 correctly electroporated in the muscle fibers leading to the expression of the 48:41 fluorescent green protein and probably also leading to the expression of the sp cast iron 48:47 and after two guide ironing we then extracted the dna from these 48:55 muscle and we first confirmed using a test called the surveyor enzyme 49:01 but indeed they were constantly being produced in exon 50 and 54. we then 49:08 use pcr to amplify the eblatex zone 5054. 49:14 note that in the muscles that are not treated with the crystal class 9 technology 49:19 there is no amplification of example 5054 because there are 160 000 base pair 49:26 between these two exiles and therefore the two primers for the pcr are too much 49:33 separated from one another 49:43 we then sequence this exon 1554 that was produced in vivo 49:50 in the mouse muscle expressing the human dystrophin gene and as predicted we had the sequence of 49:57 exon 50 and a new codon at the junction side 50:03 followed by all the correct codon coding for the normal amino acid in exon 50:09 54. this is exactly as predicted in vivo 50:17 however for delivery of the cast 9 gene in evil to several muscles 50:24 we need to use an adeno associated virus as mentioned before the sp cas9 50:31 gene is too big to be delivered with two guide rna by a single aav and we therefore use for 50:38 our next experiments the cast knife of the staphylococcus moleus which is a smaller cas9 which 50:46 permit delivery with two guide irony by a single avp in this case we have used uh 50:55 guide rna that induced cuts in exam 47 in a sequence coding for 51:01 lxb and in exon 58 in a sequence again coding for elix b 51:08 this resulted in the formation of an ebola exam 51:13 4758 coding for an ibrigid lxb which has a normal structure 51:19 the beginning of the exp be encoded by exon 47 and the end of that evil 51:27 philips be being coded by exon 58 51:34 we then use for our imbibo experiment a new mouse model called hdmd delta 52 51:43 it's the same mouse model as previously that contains the dystrophin gene with all the 51:50 exons and all the intron except that this mouse has a deletion of exon 52 and thus 51:57 there is no expression of the human dysphoric gene we have just used the crispr class 9 52:03 technology to induce a cut in exot 47 and a cut in exam 58 52:09 producing the ibrine exam 4758 resulting in the production of a 52:15 dystrophin protein containing an evil exam every lxp 52:26 we have injected to these hdmd delta 52 mouse 52:31 aevs coding for sc 9 and 2 single guide rna a month later we observe in the muscle 52:39 the expression of the human dystrophin gene including in the heart 52:47 we are just proposing a treatment of duchenne muscular dystrophy which would be the systemic delivery by 52:53 an adeno associated virus of the class 9 gene and of two guide rna 52:59 to form any bread exam in contrast with exon skipping which is a treatment 53:05 done at the level of the messenger rna the treatment that we propose at the level of the dna would be 53:12 permanent the crispr cas9 technology is evolving rapidly 53:18 and new technologies derived from the crispr cas9 permits now the modification of a single 53:26 nucleotide more than 32 000 single nucleotide 53:32 modifications are responsible for early steady disease and thus the 53:38 capacity to correct a single nucleotide would provide treatment for 53:44 most of these healing steady disease the first treatment which permits the 53:50 modification of a single nucleotide was developed by kamal in 2017 which use 53:58 a cas9 nikkei that is the modified cast 9 which is able to cut only one 54:04 strand of dna and this cast 90 case is fused with 54:09 acetaminophen this technology is permanent to 54:15 clinically modify city bean into a uvd which is replaced 54:21 by a timing on the dna 54:26 the main limitation of that technique is the chemical modification of the city 54:32 will occur in a narrow window located at 12 to 16 base pair from the 54:40 ngg pan we have initially used base editing 54:48 technology to develop a treatment for alzheimer's disease 54:57 the alzheimer's disease is produced by an abnormal metabolism of the ammunition 55:04 protein normally this protein is cut by the alpha secretaries 55:10 followed by a cut by the gamma cell this proteins peptides and protein 55:16 fragments that are degraded without causing any problems however this protein may also be cut by 55:24 the beta sequence followed by a cut by the gamma sequences and this produced 55:30 short 40 42 amino acid long beta amigo infections that aggregates to 55:37 one another forming amino acids that interfere with synaptic transmission 55:44 leading to neuron death and the memory problems 55:50 this scheme illustrates the amino acid sequence of the transmembrane part of the 55:56 anaerobic repressor protein we can see the position of the beta alpha 56:02 and gamma separates calcite all the amino acids in the star above 56:08 their main are amino acids which are modified leading to 56:13 family or form of alzheimer's disease please note the position with the red arrow this is the alanine 56:20 in version 673 when this adenine is changed by valley 56:25 this leads to severe early onset alzheimer's disease and you 56:30 are a zemer at the age of 40. however when this adenine is changed by a journey 56:37 you're not alzheimer even when you're 95 105 years old as shown by johnson nepal in nature 56:44 2012. 56:49 the presence of the a673t mutation also known as the icelandic 56:57 reduce mutation secretion of a beta 40 in our data quality peptide 57:02 for the wild-type appg and for appg containing the london mutation 57:12 our experiments have shown that the presence of the h673t mutation 57:20 reduce the secretion of a beta 40 and a beta 42 peptide by the epp 57:27 genes not only for the wild-type gene but also for app genes containing several familial 57:34 alzheimer's disease mutations 57:41 as mentioned before the crispr cas9 base editing technology permit to modify 57:48 the city dean into a timing 57:55 we just have used the base editing technology to target the cytoplane in the 58:03 antecedent strand of the alanine codon transforming that cytodine into a timing 58:09 and just changing the alanine codon into a triangle 58:18 the main problem with this approach is that although we want to modify the cytodine 58:24 into the ents decent strand of the adenine codon there are other acidity nucleotides 58:32 nearby that are also affected by the base editing approach 58:40 we have constructed 14 different base editing enzymes to be able to quantify 58:47 more specifically 58:56 by modifying the city and the antisense codon of led we have been able to introduce 59:05 mutation in up to 17 of the avpg however other acidity 59:13 also located in the antecedent strand were also modified by the subject 59:22 a new fantastic technology called privacy has recently been developed by pencil on 59:28 top with permits in principle 59:38 the prime editing technology use a cas9 new case fuse with 59:44 a reverse transcriptase it also requires a prime editing guide rna known 59:51 as a pig rna 59:57 the pig rna is essentially a prolonged single guide rna thus has a single guide 1:00:04 rna it contains a spacer sequence which react with 20 nucleotide 1:00:11 in the target dna it also contains the constant scaffold of the single 1:00:17 guide rna which is in red and then at its 1:00:22 five prime end there is a prolongation with the primer bending side which is a 1:00:28 sequence of 10 to 17 nucleotide reacting with the upper 1:00:33 strand of dna this is followed by the reverse transcriptase template 1:00:39 again which is 10 to 17 nucleotides in length and which will contain some modified 1:00:46 nucleotide in red in this case indicating which nucleotide 1:00:52 has to be modified by the reverse transcriptives 1:00:59 a plasmid designed by anzalo natal 1:01:04 is available at edgy to construct new peg rna 1:01:14 this plasmid contained a red fluorescent protein gene which may be removed by bsa1 cuts which 1:01:21 produce a backbone of the protein and then the other components are the spacer primer binding side the reverse 1:01:27 transcriptase template and the pig rna scalp all of these sequence are single 1:01:33 stranded polygon nucleotide that may be purchased from idt these four parts are then assembled 1:01:40 together to produce a new pay guarantee 1:01:46 to use the prime editing technology we first have to identify photo spacer adjacent positive tab 1:01:54 which is njj for the class nine of streptococcus pyrogene 1:02:00 this will permit to the cas9 to bind to the dna then the spacer sequence of the peg rna 1:02:07 will bind to a 20 nucleotide sequence of dna in this case lower strand and the formation of the 1:02:14 complex between the peg rna class 9 decades and 1:02:19 the dna will induce the nick at exactly 3 nucleotides from the path 1:02:25 in the upper strand of dna this will release the upper strand of dna to be able to 1:02:33 interact with the primer binding side of the pile the peg rna and then the reverse 1:02:39 transcriptase template which contained a few nucleotides to be mutated will be available for the 1:02:47 reverse transcriptase to synthesize a new dna 1:02:52 upper strength meaning duchenne muscular dystrophy 1:02:59 patient have a stop called on the dmdg since we did not have 1:03:05 access to cells of patient containing such pointation we decided to introduce 1:03:12 these stop codons using the prime editing technology so in each case we had to identify 1:03:19 an ngg pam identify the peg rna photo spacer sequence and then 1:03:27 modify the reverse transcriptase template so as to enter to modify the 1:03:33 codon for an amino acid into a stop codon in this case we have changed 1:03:41 acetaminophen to introduce the tga stuff 1:03:50 we have successfully used that approach to introduce stockholders in exam 9 20 35 1:03:57 42 55 and 61. 1:04:04 we have just designed various peg rna targeting dmd exon 35 as you can see we have 1:04:12 varied the reverse transcriptase template in blue from 10 to 16 nucleotide 1:04:20 and we have also varied primary binding site in green from 10 to 15 nucleotides 1:04:27 and at the desired mutation site we have introduced a t to 1:04:34 introduce that mutation 1:04:41 our initial experiments were done in hek293t cells 1:04:48 we tried to reproduce the mutation of mx1g and to target 1:04:55 dmd exon 35. so essentially these cells were transfected 1:05:01 with plasmid coding for the cas9 djs fuse with the reverse transmitted days 1:05:07 and a paid rna targeting either exon 35 of the dmd gene 1:05:13 or the e mx1 g the dna was extracted three days later 1:05:20 and the targeted sequence was pcr amplified and sequenced using center method 1:05:26 the sequence were analyzed using the edit r online program essentially we observe 1:05:34 a 32 percent correction mutation of the air x1 gene has 1:05:41 done by heads along at all however for the exon 35 mutation 1:05:48 we had a two percent background in the sequence of the control negative 1:05:55 control and with the different peg rna we have mutation ranging from four to eight 1:06:02 percent so this was not as great as for the e m x one g 1:06:13 we have just tried different method to try to increase the percentage of genome editing 1:06:18 of exon 35. the first method that we have tried is to repeat the treatment three times 1:06:26 essentially the cells were transfected with plasmid at the 0 6 12 1:06:32 and dna was extracted three days and six days after each treatment 1:06:40 we have amplified the pcr exon 35 at each of the extraction 1:06:46 date and sequence it by center and analyzer sequence as you can see 1:06:52 the percentage of genome editing increased from day 3 to day 18 with the 1:06:59 treatment and this was the case for all three peg rna network targeting exon 35. 1:07:11 we have then tested a second method to try to increase the percentage of mutation in exon 35. 1:07:18 it is to induce a second nick in the target g this is the pe3 method 1:07:25 essentially we have identified two pam sequence which permitted to kite rna to induce 1:07:32 a second nick at either 57 nucleotide from the original 1:07:40 big irony neighbor 1:07:54 24 nuclear time or at 57 nucleotides induced by the pig 1:08:02 there was a significant increase in the addition of the target gene propaganda 35 1:08:09 four and figure eight by six but not for bigger only 20.5 1:08:15 you still have to understand why 1:08:22 i method to improve the percentage of mutation in the target gene is to mutate 1:08:29 the pam used by the peg harmony 1:08:36 we just designed pig rna that were not only able to introduce a stop codon 1:08:43 mutation but we're also able to mutate simultaneously 1:08:48 the pan and the use 1:08:54 mutation of the pan used by the peg rna improve the percentage of mutation in 1:09:01 the stop codon for two of the three peg rna that we 1:09:06 have tested 1:09:12 combining the two method that is inducing a second nick in the target g 1:09:19 and mutating the pan used by the peg rna further increase the mutation of dmd 1:09:26 exon 35 to 39 with all three pegs that we have 1:09:32 tested 1:09:41 we are currently starting new project to correct mutation responsible for other heading steady 1:09:47 disease we are working on cystic fibrosis due to mutation in the cftr chloride channel 1:09:55 on congenital muscular dystrophy due to mutation in the rheanodine receptor and on 1:10:02 ataxia 8 due to mutation in the nkx6 1:10:07 type 2 gene 1:10:13 for each hearing steady disease due to a point mutation for example here with attacks at 1:10:19 type 8 it is possible to correct in principle the mutated gene using the prime editing 1:10:26 technology in this case here we can identify that the mutation is an adenosine change for timing 1:10:34 and thus we can identify a pan ng for the sp cas9 which is close to the mutated 1:10:40 nucleotide we can then design a plane rna that will introduce the desired mutation in this 1:10:47 case we are introducing two mutations one to correct the mutation 1:10:53 to reverse the timodine into an adenosine and 1:10:58 the second midpoint mutation is to modify the path so that following the 1:11:03 correction the cation enzyme can no longer bind to the dna 1:11:13 crisper casino derived technologies may not be used to treat many different city 1:11:18 diseases the main problem remained the inhibit delivery of the editing agents 1:11:27 thank you for your attention 1:11:35 okay thank you professor jax i'll take the turn now to make a general comment for those who 1:11:44 the who doesn't speak uh those who don't speak english okay thank you for your 1:11:49 presentation thank you of [Music] 1:12:00 [Music] 1:13:17 i 1:14:04 [Music] 1:15:29 [Music] 1:15:43 [Music] 1:15:52 [Music] 1:16:08 [Music] 1:16:32 foreign 1:16:59 [Music] 1:17:42 foreign 1:17:51 [Music] 1:17:57 [Music] 1:18:10 a [Music] 1:19:25 he summarizes the your speech okay not the song 1:19:39 yes sir thank you very much for the the translation of my summary in portuguese 1:19:47 okay um 1:20:02 foreign 1:20:25 [Music] 1:20:30 is 1:21:12 gabriel 1:21:39 thank you for having me uh let's see if we can get it started 1:21:52 yeah uh can you see it hi my name is and today i'll be discussing 1:21:58 the detection of stars using crystal technology named the test that we've been 1:22:03 developing and dr tremblay's Hospital studies have confirmed that many individuals infected with 1:22:10 earth ob2 virus or asymmetric carriers 1:22:15 you can't hear yet no yes can you hear it 1:22:23 no just maybe you should talk 1:22:31 okay uh i can actually try streaming it one other way um if that's the case so 1:22:50 okay 1:23:02 hi my name is gabriel lamot and today i'll be discussing the detection of cyrus kovi 1:23:09 yeah that's much better is the sound better now e2 using crispr technology oh perfect 1:23:14 namely the test that we've been developing in dr trombley's laboratory studies have confirmed that many 1:23:20 individuals infected with osiris-cov2 virus are asymptomatic carriers of the virus 1:23:26 this is problematic in that the pandemic might be growing without adequate supervision simply focusing the testing on 1:23:32 individuals with symptoms as opposed to entire populations is not enough 1:23:37 to better contain the virus until a large-scale distribution of an effective vaccine is underway large-scale and 1:23:43 continuous testing is mandatory the current diagnostic pipeline for 1:23:49 detecting sars cov2 is a three-step process the first step is to obtain nasopharyngeal swabs from 1:23:56 patients the next step is to isolate the total rna from said samples and provide the rna required for this 1:24:03 third step which is to use real-time quantitative vcr machines the rna is first reverse transcribed 1:24:10 into dna and then amplified a dye is added to the amplification reaction to signal the presence of dna 1:24:15 strands after a certain number of amplification cycles a positive result will give 1:24:20 a strong enough signal that the machine flags it as containing the original virus now this process is problematic for a 1:24:28 few reasons the first is that real-time pcr machines are extremely expensive 1:24:33 most can range from fifteen thousand us dollars to well over ninety thousand us dollars 1:24:40 given that it's a fairly specialized piece of equipment and that not all molecular biology laboratories need one some get 1:24:46 along just fine with a regular pcr machine which costs a much more reasonable 5000 us dollars 1:24:52 as such widespread testing for the coronifiers using these machines isn't ideal 1:24:57 acquiring the large number of machines required for widespread testing is a feat in and of itself 1:25:03 and is likely one of the contributing factors for the sometimes long wait times for results 1:25:09 many reports have stated that it can take 3 to 24 hours or more to obtain results in a clinical 1:25:15 diagnostic laboratory over here in canada ontario reports wait times of up to four days to get results 1:25:22 high density metropolitan areas like new york are also subject to long lines for testing and then a time to 1:25:29 results of about seven days for some clinics in that city the creation of new types of detection tests 1:25:36 is therefore an important step in curbing the spread of this virus an ideal test needs to follow the world 1:25:43 health organization's criteria for assured that is the test needs to be affordable 1:25:48 for as many people at risk of infection as possible tests that require extremely expensive reagents or machinery 1:25:55 are not ideal the test must also be specific to avoid false negatives that way anyone 1:26:01 who could potentially spread the virus can be identified and put into quarantine the test must be sensitive so 1:26:06 that few people who don't have the virus are mislabeled as having it and treated improperly the 1:26:12 test must also be user friendly that is it must be simple to perform and it must be rapid and robust 1:26:18 that is it must give a time to results that is quick enough to make the most use from the results 1:26:23 situations where it can take up to seven days to obtain results are clearly far from ideal as by the 1:26:29 time the patient receives the information they might have infected many others 1:26:34 the tests must be equipment free as much as possible tests that require too many rare and expensive machines aren't ideal for many 1:26:41 countries finally the test must be deliverable to those who need it 1:26:48 the discovery of cast 13 or c2 c2 was revolutionary in the field of molecular detection 1:26:54 this class 2 type 6 crispr effector is similar to the protein cast 9 off of 1:27:00 which base ending and prime ending were designed this protein is also a member of the crispr family 1:27:06 unlike cas9 however cast 13 can be programmed to target rnas as opposed to dnas 1:27:13 by binding to its crispr rna cast 13 is primed to target extremely specific rna sequences after locating a target 1:27:20 sequence the cast 13 protein is activated this causes the protein to begin to cleave 1:27:26 everything its surroundings in an event called collateral cleavage it cleaves its original target 1:27:32 and then moves on to start cleaving all surrounding rnas researchers realized that this 1:27:37 collateral cleavage could be used to turn the protein into a switch for a detection test once 1:27:43 the target molecule is present the switch is flipped the cas13 activated and all surrounding rnas are degraded by 1:27:51 tagging certain rnas in the solution with a fluorescent molecule and a quencher 1:27:56 it would therefore become possible to resolve a signal researchers therefore started to develop 1:28:02 a detection test based on this protein for viruses bacteria and human diseases such as cancer 1:28:09 however they soon noted that there were certain limitations to the protein and that his limit of detection was far from 1:28:14 sensitive enough for their needs recently certain tests have been designed using only cast 13 1:28:21 however these tests are less sensitive and tend to use a different cast 13 protein 1:28:27 researchers at the broad institute decided to supplement the protein with a few others that would amplify the 1:28:32 target rna to this end dr zhang and his team created a new test called specific high sensitivity 1:28:39 enzymatic reporter unlocking or sherlock for short this test is based 1:28:45 on the reverse transcription of target rna into dna afterwards the dna is amplified at a 1:28:50 constant temperature by using a process called recombinase polymerase amplification 1:28:56 this technique amplifies a small amount of dna at a constant temperature unlike the more commonly used pcr 1:29:03 the incorporation of this step also means that the ensuing detection test can target dna since the rpa reaction 1:29:10 simply amplifies a determined section of dna using two primers the initial genetic sequence can either 1:29:16 be rna or dna so long as reverse transcriptase enzymes are present for rna 1:29:24 the amplified sequences are then transcribed using a t7 rna polymerase to create vast quantities of the target 1:29:30 rna this then allows the cas13 to have a far larger pool of target molecules 1:29:36 which can activate the enzyme once the enzyme is active it does what we discussed before and 1:29:42 starts to cleave all surrounding rnas including reporter rnas which give off the easily detectable signal 1:29:48 which makes the test work in their initial version of the sherlock test two different reporter 1:29:55 molecules were used the first used a fluorescent molecule as well as a quencher 1:30:00 and it was able to produce a lot of fluorescence after cleavage of the rna the other 1:30:08 was designed to interact with a few antibodies and when it was cleaved it appeared as a distinct 1:30:13 second band on a strip that was quite similar to a pregnancy test 1:30:18 while this test was good in many ways it had certain drawbacks at the beginning of the pandemic we attempted to replicate it and 1:30:25 immediately had a few issues the biggest issue by far was that some of the materials required for the test 1:30:30 to work were only available in extremely limited supply the kits required to perform rpa 1:30:36 reactions for example took approximately two months to arrive and even then we only received enough 1:30:41 materials to perform 200 tests this clearly wasn't going to be enough to produce sufficient tests in the 1:30:47 future additionally the reporter that used a pregnancy style strip was remarkable and seemed to be very 1:30:54 useful however since it was too difficult to acquire insufficient quantities 1:30:59 we decided not to use it and to instead focus our efforts on using the fluorescent style reporter rna since the original version 1:31:07 of the test could not be used by us in the way that it was intended we decided to redesign it 1:31:14 in redesigning the test we had several options and routes that we could take we knew that we wanted to keep on using 1:31:20 cast 13 because we had a large supply of this enzyme due to an ongoing collaboration with dr 1:31:26 alain yani's lab we also knew that we wanted at all costs to maintain the isothermal nature of 1:31:32 this test one of the biggest advantages to sherlock was that it didn't require large amounts of expensive equipment 1:31:38 simply by using a heating block or something of the sort it was possible to get a visual readout 1:31:44 we therefore decided to employ loop-mediated isothermal amplification or lamp for short this amplification 1:31:51 strategy was fairly well documented and easy enough to perform based on the use of six primers 1:31:57 this technique could rapidly amplify large amounts of target dna unlike both rpa and pcr however lamp 1:32:03 doesn't create identical amplicons of a discrete size instead the six primers can interact to 1:32:09 create far larger molecules of a repeating sequence initially it was a little difficult to 1:32:15 incorporate this isothermal amplification strategy into our test as you might remember the step following 1:32:21 rpa in the original sherlock test was one that produced lots and lots of target rna 1:32:27 this production of target rna was only possible because the rpa step added a tag that let the t7 rna 1:32:33 polymerase bind to the dna and begin transcribing it however no one had seemingly ever done 1:32:40 that with lamp and since this new technique used six primers two of which looped back on themselves to create 1:32:45 weird dumbbell-like structure structures it was difficult to figure out exactly where to incorporate this tag 1:32:52 after a while we decided to include the tag right in the middle of one of our looping primers this has consistently 1:32:59 given us very strong results we were therefore able to retrofit the original sherlock 1:33:04 test with a new amplification strategy all the while still benefiting from cast 13's specificity and signaling 1:33:12 the new test is therefore using rt lamp as opposed to rt rpa of course 1:33:18 should we become interested in targeting dna in the future for either certain types of viruses or 1:33:24 certain bacteria this test would be readily applicable to that as well we also decided to use a fluorescent 1:33:30 reporter rna instead of the pregnancy style one that we spoke about previously because it seems to be the most readily 1:33:37 available material since it doesn't take any particularly advanced machinery to make it work 1:33:42 we were happy to choose this one 1:33:48 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 1:33:54 the test requires that we start with rna that was extracted from a virus using a commercially available kit one 1:34:00 microliter of this solution is transferred over to the tube containing the rt lamp reaction 1:34:06 ideally in the future we would like to modify the test so that it becomes possible to directly add a patient's saliva 1:34:12 to it and continue on from there that would noticeably cut down on the labor required to obtain each reading 1:34:18 cut down on the overall cost and make it much faster for the results to come in 1:34:23 after transferring one microliter of the rna extract to the first tube the tube must then be incubated at 65 1:34:29 degrees celsius for 30 minutes after that one microliter is transferred from the first tube into the second 1:34:36 which contains the t7 and cast 13 solution after tapping the tube a few times to 1:34:42 mix it and microcentrifuge it down for a few seconds all you need to do is incubate that tube at 37 degrees celsius 1:34:49 for 30 minutes at that point all of the reactions are done and that the results are ready to be 1:34:54 read by exposing the tubes to a wavelength of approximately 490 nanometers it's possible to easily 1:35:00 detect which samples contain cyrus kov2 those ones will fluoresce a bright green 1:35:07 those that didn't contain any of the viral specimens of interest will be completely colorless and 1:35:12 indistinguishable from a tube filled with a little water i'll show you that later 1:35:18 now it's worth noting that the dna and rna sequences that we have used in this first version of our test 1:35:23 were all previously published by different groups as such we do not believe that they will 1:35:28 result in a positive signal when in the presence of a relevant rna 1:35:33 this is particularly important because your saliva is full of rna the extraction process for the virus 1:35:40 does indeed indeed result in viral rnas being captured but there tends to be a whole lot more 1:35:45 human and bacterial rnas too in addition other coronaviruses that are 1:35:51 much less dangerous are fairly common in the human population if our test gave positive signals every time someone came in 1:35:57 with a common cold then it wouldn't be very effective as such we have based ourselves on 1:36:02 previously described templates to try and avoid this as much as possible 1:36:08 in designing this test we tried to make sure that it wasn't overly reliant on complicated machines 1:36:14 we wanted the entire test to be easy to perform using only a few common laboratory apparatuses 1:36:20 as a result with this current iteration of the test all we need is one machine that can heat 1:36:25 pcr-style tubes to 65 degrees celsius and one that can heat 1:36:30 the pcr tubes to 37 degrees celsius we also need two either p2 or p10 1:36:37 pipettes for reasons we'll get into later along with their associated pipette tips 1:36:43 a small microcentrifuge capable of taking pcr tubes is also required just to make sure that the reaction 1:36:49 mixtures don't touch the top of the tube when you're opening them and potentially contaminating all of the 1:36:55 surrounding tubes finally a machine capable of giving off ultraviolet wavelengths is necessary 1:37:02 in this picture you'll see the machine i've been using it's most commonly used to visualize gels in a molecular biology 1:37:08 lab but it does a really good job of visualizing our positive samples in our test for the two incubation 1:37:15 periods several different options are available i've been using an old thermocycler today 1:37:21 as i find it does a really good job of evenly heating my tubes it's worth noting here that this means 1:37:26 that even old and run-down thermocyclers that no longer do a very good job of cycling between temperatures 1:37:32 can be readily used in this test unlike the current rtq pcr gold standard that requires 1:37:40 specialized real-time thermocyclers here any old machine can be used 1:37:45 it's also possible to use other common laboratory apparatuses such as block heaters that have an 1:37:51 adapter for pcr tubes please note that in this picture the block heater has an adapter for 1.5 1:37:57 millimeter microcentrifuge tubes so the true adapter will look a little different 1:38:03 i've also used incubators to great success even material incubators that operate at a steady 1:38:08 37 degrees celsius can perfectly suit the needs of this test so long as the tubes are readily exposed 1:38:15 to the ambient temperature then positive signals should have no difficulty in resolving 1:38:20 please note however that when using incubators it's better to have the tubes exposed to the air 1:38:25 that way they don't waste time as their rack slowly begins to heat up 1:38:31 water baths are one final option that you can choose personally i tend to avoid this option 1:38:36 because i find the risk for cross-contamination between samples to be much greater and therefore unideal 1:38:43 however i have tried to perform rt lamp reactions with this system and so long as you're 1:38:48 careful it's very possible to do it 1:38:53 once the reactions have been completed it's possible to use various different machines to visualize the tubes 1:38:59 depending on the materials your laboratory has personally my machine of choice is a gel 1:39:04 uv emitter these machines which are more commonly used to visualize agarose gels 1:39:09 do a very good job of safely showing which tubes are fluorescent and which aren't black lights can also 1:39:16 be used for this test however i have found them to be slightly less desirable since the reporter rna best absorbs 1:39:23 wavelengths of 490 nanometers i have found that black lights which produce wavelengths of 390 nanometers 1:39:30 to be less favorable for this test for them to work it's better to visualize the tubes in a dark room 1:39:37 in both cases the machine should really be placed in a darker room away from excessive light a little 1:39:43 background light seems to have less of an effect in the case of the gel uv light however 1:39:48 this picture seen on the left was taken using a generic iphone when the tubes were placed on the gel uv 1:39:55 light the lights in the room were only partially turned off resulting in lots of ambient lighting 1:40:01 even if there were no lights directly pointed at the machine given the strong and even radiation that the tubes 1:40:07 received on this machine it's easy to determine which samples were positive and which were negative 1:40:14 in the case of the black light on the right it's clear which samples are positive and which are negative 1:40:19 of course however the fluorescence is not even and it's a little bit harder to detect 1:40:24 by the naked eye in this case the camera on the iphone took a very good picture 1:40:29 and made it easier to visualize the tubes overall now there are several important 1:40:35 considerations to be had when using this test for it to work properly and not give either false positives or false 1:40:41 negatives it's important to first designate proper working stations for each section of the test it's also 1:40:48 necessary to follow the predetermined reaction times without varying otherwise the results might start to differ 1:40:54 slightly finally those handling the test must be careful not to contaminate any of the tubes with 1:41:00 rnases the first consideration to separate working areas 1:41:06 is extremely important because lamb is excessively sensitive this technique is known to produce lots 1:41:12 and lots of concentrated target dna we as well as others have noted that 1:41:17 when we opened tubes that completed the lamp amplification in the same area that we originally used 1:41:23 to set up the reaction it's possible to contaminate the working area with aerosols containing the 1:41:28 product if you continue to set up the lamp reactions in those areas afterwards then you run the risk of 1:41:34 contaminating your new tubes with amplicons from the previous reactions by touching your desk or 1:41:40 various other contaminated surfaces with your gloves you run the risk of artificially seeding your reaction 1:41:46 mixtures that contain patient rna with no sars cov2 and therefore producing positive results 1:41:52 when there really shouldn't have been any by separating the areas in which you set up the lamp reactions 1:41:58 and those in which you use the amplified sequences you run less of a risk for this to happen 1:42:04 it's also necessary to have designated lab coats pipettes and other such materials for each 1:42:09 working station if those in workstation 2 become contaminated you don't want that coming 1:42:14 back into workstation 1. please note that this is much less stringent when bringing materials from 1:42:20 workstation 1 to workstation 2. since it's the rt lamp reaction that 1:42:25 runs the greatest risk of contamination bring material for example gloves 1:42:30 from that station to the next should not be a problem provided that the gloves are still 1:42:35 relatively clean please note that this situation of contamination via aerosol 1:42:42 is of much less importance during the second stage of the test as we discussed previously the cas13 1:42:48 protein we are using requires a relatively large amount of rna to be functional 1:42:53 at the present time we are not concerned in the least that an aerosol contamination from lamp 1:42:59 might produce false positives of our cast 13 reactions as we have yet to see a single situation 1:43:05 where this has happened the second consideration of note is that 1:43:10 the reaction time must be followed a small discrepancy can be tolerated that is one minute more or less doesn't 1:43:18 really seem to have a large impact on the test however more than that can start to become problematic 1:43:24 in the case of the rt lamp reaction external teams as well as us have seen that by doubling the reaction 1:43:30 time for the lamp false positives can arise as you can see here in both pictures 1:43:36 a successful lamp reaction can also be visualized on an agarose gel the successful reactions are 1:43:43 characterized by the presence of several large bands of varying sizes however when the lamp 1:43:49 reactions go on for too long it's possible to begin amplifying samples with nothing in them 1:43:54 for example on the left you'll see a gel that was taken from an article in which at 30 minutes the wells 1:44:01 containing reactions with 10 copies of the target are viral rna and no copies of the 1:44:06 target viral rna had no bands in the case of the gel on the right a gel that i made three of the wells 1:44:13 contained reactions that had no target rna to speak of only the n fragment well contained 1:44:20 target rna at 30 minutes these negative controls did not demonstrate any amplification 1:44:26 as we expected however at 60 minutes both the published gel and mine started 1:44:32 to give amplified products in their case the 10 and 0 copies both 1:44:37 started to be amplified and in my case one of my negative samples started to get amplified i've performed 1:44:45 several experiments so far with 30 minute incubation times and so far have never gotten false positives that's 1:44:53 not to say that it's entirely impossible simply that it's very very unlikely 1:44:58 as a side note this is a good time to mention that changing gloves frequently when performing the first reaction is a 1:45:05 good way to avoid these kind of unlikely false positives after prolonged use 1:45:10 gloves might become contaminated with small amounts of viral rna and that could potentially lead to false 1:45:15 positives even if we're only incubating at 30 minutes 1:45:20 the final consideration to look out for is the presence of rnases for those of you who might not be 1:45:26 familiar rnases are enzymes that cleave all rnas they come across 1:45:31 both steps for this test are sensitive to rnases the initial arty lamp begins its 1:45:36 amplification off of rna so the presence of rnases could be very detrimental and therefore 1:45:42 lead to false negatives the second step of the test the t7 and cast 13 step uses an rna 1:45:49 reporter to signal positive tubes if while you're using the tests you start to introduce rnases 1:45:55 then you run the risk of getting false positives rnases are extremely difficult to remove 1:46:01 once they're introduced into a system so the best means of protecting your test is by using prevention 1:46:06 since your hands produce lots of rnases it's mandatory to use gloves at all times when working with the test 1:46:13 in doing so you will prevent your body's natural rnases from degrading the initial stars cov2 1:46:18 rna and protect the fluorescent reporter at the end gloves must also be changed whenever 1:46:23 they come into contact with skin hair or other frequently manipulated objects 1:46:29 such as doorknobs and personal devices the use of rnas and dnase free 1:46:36 filtered pipettes is also important for this test by using these kinds of specialized tips 1:46:42 you can make sure that any possible contaminations you might have in your pipettes doesn't have a chance 1:46:48 to contaminate your test in areas where the risk for rnas contamination is fairly high it might be 1:46:54 worthwhile to treat all surfaces or tools with an rnas removal solution 1:46:59 these kinds of solutions can degrade rnases that might otherwise stick to your gloves and contaminate your samples 1:47:06 please note i haven't had to use the solution very often so long as you maintain a clean working 1:47:13 schedule you should be fine the second component for the test the one containing t7 and cast 13 is 1:47:21 also designed to be able to tolerate a certain level of contaminating rnases there is an rnas inhibitor added to the 1:47:28 reaction mixture to prevent the degradation of the reporter in case the original sars kobe 2 rna 1:47:35 wasn't completely clean or in case contaminations occurred while you were repairing the tests 1:47:41 that said the presence of excessive rnases can still fully activate the test seen 1:47:48 here on the right one microliter of very concentrated rnase a is capable of causing the test tube to 1:47:54 become fluorescent it's therefore important to take the 1:48:02 required 1:48:16 cabriolet 1:48:26 test that can be widely distributed while also meeting this limit of detection is fully functional for curbing the 1:48:34 spread of this pandemic while rtq pcr is much more sensitive 1:48:39 than this its improved limited detection might not necessarily be that beneficial 1:48:44 since this technique can pick up rnas freed by infected and dead cells after the infection has run its course 1:48:51 this form of screening can cause individuals who are nominally sars kovi to rna 1:48:56 positive but aren't actually infectious to be isolated unnecessarily 1:49:02 as you can see here on our left our detection test is able to detect samples 1:49:07 that have 100 copies per microliter as was stipulated by the model a more 1:49:12 recent comparison between the different primers that we've been using for lamp has demonstrated 1:49:17 that one version of our test can detect samples containing only 80 copies per microliter 1:49:23 while the other can only detect samples containing 1000. moving forward we're likely to focus our 1:49:29 efforts on the first primer set to ensure that we meet the limit of detection that was 1:49:34 stipulated by the model as a final note we're happy to report 1:49:39 that our test works not only on fragments of the cyrus cov2 genome it also works when exposed to the 1:49:46 virus's full rna profile initially we began the development of our test by using fragments 1:49:52 of the viruses genes that we had transcribed ourselves in the lab recently we've used the test 1:49:59 on actual sars cov2 viruses and have obtained the expected results 1:50:04 as you can see on the left the first section of the reaction the rt lamp reaction works just as 1:50:10 expected on the viral rna the top left wells that were run on the agarose gel clearly demonstrate 1:50:16 a strong amplification below them three reactions were performed with cells that had not been cultured 1:50:22 with the virus the viral rnas were created by first isolating the viruses from a patient 1:50:28 these viruses were then cultured in vero e6 cells and after a few days the culture medium 1:50:34 was treated to extract any and all rnas that were present the viruses were therefore destroyed and 1:50:40 their rna was obtained when the lab amplicons that had amplified the real virus 1:50:46 were transferred over to the second stage of the test they gave the expected strong fluorescent signals 1:50:52 at the moment what remains to be done is to use our tests on samples that have been confirmed positive or 1:50:58 negative by rtq pcr the current gold standard in sarsko v2 detection 1:51:05 and see how our test compares this will give us a better indication of what our tests sensitivity and 1:51:11 specificity are just to wrap up i would like to thank dr gary cobinger 1:51:17 dr alain ghani and dr ghiblave for having helped to provide finances or materials 1:51:23 for this project i'd also like to thank dr tremblay and everyone in his team for having helped to provide certain 1:51:29 insights as i was redesigning this test finally i'd like to thank dr marcelo for 1:51:35 having helped to organize this entire meeting and the canadian institute for health research 1:51:40 and the frqs for having helped finance me as i pursued this project 1:51:50 okay so that was my presentation uh yeah 1:51:58 thank you gabriel uh i'll take the time just to make a to summarize it the best i can here for 1:52:05 the non-english speakers thank you very much for the presentation thank you okay then 1:52:13 [Music] 1:52:34 [Music] 1:52:40 [Music] 1:53:10 is 1:53:24 is 1:54:06 foreign 1:54:20 positive 1:54:50 [Music] 1:55:34 [Music] 1:56:06 [Music] 1:56:30 [Music] 1:56:41 [Music] 1:57:04 [Music] 1:57:32 [Music] 1:57:38 thank you matilda and thank you dr graviol 1:57:44 professor jackson do you like to talk something at this point uh i have nothing to add 1:57:50 except if there is some question that i can answer otherwise i thank you for organizing this session 1:58:00 thank you question 1:58:05 is uh writing english can you read the the commentary first 1:58:11 one can you read the comments yeah but i think that most of them 1:58:17 are in portuguese no i asked you to write in english i 1:58:23 have in the comments oh okay i was looking at private chat 1:58:34 i'm going up the list oh boy lots of 1:58:46 comments 1:58:54 are you in commencement chat yeah i'm just looking at the comments very quickly 1:58:59 uh grow up anachrestia is is is the probability 1:59:06 that the crispr test will give false negative results that's a question mostly for gabrielle gabriel 1:59:14 can you answer that uh so at the moment we don't entirely 1:59:20 know with my test generally um 1:59:25 tests similar to this one have a sensitivity of about 90 percent so the probability of giving 1:59:32 a negative result is very low because it also tends to have a sensibility of about 80 to 90 as well so overall it's better 1:59:41 than some of the alternatives that exist that aren't rtq bcr another question for 1:59:46 you it's also from anna christina does the crisper test 1:59:52 apply to other disease like ebola zika or dank 1:59:59 so we actually intend to adapt the test to ebola uh as soon as we can um we're toying 2:00:05 with the idea of doing zika leps and a few others because all we need to do is change a few of the 2:00:11 sequences that we're using so the lamp primers and the guide for the crisper cast 13 protein and at that 2:00:18 point we'll immediately be able to start detecting those viruses as well so now the test exists it's just a 2:00:24 matter of optimizing it for every individual virus okay there's another 2:00:30 question from france antarctic nouvelle uh what are the perspective of using the crispr 2:00:35 castline technology to treat hiv i must admit i'm not 2:00:41 an expert of hiv i know that some groups are working on that i don't know what are the chance that 2:00:48 whether or not this will work but i i think this is very zonable you know the bacteria 2:00:54 have been using the crispr cat9 technology since millions of years to cut the virus and so the idea here is 2:01:02 to use the cascade technology to crush to cut the hiv virus 2:01:07 i think this is quite notable in fact at the beginning of the cobia 19 pandemic i saw a group in the united 2:01:15 states that were proposing to to use the crispr cast 9 technology 2:01:21 indeed to cut the copy 19 virus so it's the same approach for the hiv i 2:01:27 think this is a reasonable approach my lab is not working on that approach 2:01:36 also from commencement uh can this technology supposed to be antibiotic in the near future for 2:01:44 example to treat multi-resistant tuberculosis again i think it's a good suggestion and 2:01:52 yes indeed the main problem with the with these crispr cas9 technology 2:01:58 is to find an adequate technique to deliver either the cast 9 protein of the 2:02:03 castline gene and so far in gene therapy what is being used is the adeno associated virus 2:02:11 unfortunately i didn't know associated virus custom fortune to produce 2:02:16 in good manufacturing practice so we have to find another delivery method to deliver into the cas9 2:02:23 protein or the cas9 gene and then it would ease using it because right now it would be 2:02:29 just too expensive to be used instead of an antibiotic 2:02:36 also from french and tactic crispr cast 9 may be an option in the 2:02:42 near future to treat koben ebola zika malaria well all of that i think 2:02:50 is feasible if i would have more money in my lab we could start working on that also i i 2:02:57 think these are a reasonable project [Music] 2:03:09 [Music] many of the comments are in portuguese 2:03:15 unfortunately there is no question for you yes only 2:03:23 comments no questions let me try to help 2:03:29 is there any questions only only command yeah 2:03:38 i see that i vary between two and five stars 2:03:46 well one question from vitor is it like to know it is i'd like to know if 2:03:52 crispr is affordable to public health system and if it can contribute to the cost 2:03:59 reducing of genetic therapy that already exists 2:04:05 the main cause of gene therapy that currently exists is the production of the adeno 2:04:11 associated virus in good manufacturing practice condition the last time i look into doing a 2:04:18 clinical trial or a systemic delivery of some gene therapy 2:04:24 you you took about a million dollar to produce enough aav to treat one patient so to do 2:04:32 a clinical trial on 10 patients you needed to just to start a budget of 2:04:37 10 million and of course so far i have not been able to obtain such large budget uh the only 2:04:45 people that have such large budget are american companies that will spend a fortune doing these 2:04:52 clinical trials and they have millions of dollars available the problem is indeed that 2:04:58 after the treatment has been demonstrated effective the cost of the treatment is 2.3 million 2:05:05 dollar per person so i i think it's it's the future we need to develop gene 2:05:12 therapy for many many study disease but we also need to develop 2:05:17 a cheap gene therapy you know i i think ideally a gene therapy should be around 100 000 2:05:25 not a million dollar because most of the health public health system cannot afford to do that i think that 2:05:32 it's only in united states where rich people can afford to pay for those 2:05:37 and poor people will not get access to the treatment 2:05:42 [Music] still looking down for the list to see if there is any other question 2:05:51 excuse me professor jax just to make a small comment here for 2:05:57 those who doesn't speak english i'll try to get the general idea from the answers you just gave and grab 2:06:05 gabriel also uh just a second 2:06:10 principle foreign 2:06:21 [Music] 2:06:42 foreign 2:06:56 technology 2:07:51 thank you for your time and if is there any question you can keep reading and answering them 2:07:59 well if there are other questions people can always send me some 2:08:04 emails okay uh thank you professor jack england 2:08:11 for this wonderful meeting thank you gabrielle thank you barbara dr barbara 2:08:19 thank you matteo shots and i hope you have another another one meeting like this 2:08:28 thank you thank you very much thank you goodbye thank you thank you have a good