MY NAME IS GIGI AND IT'S MY PRIVILEGE TO INTRODUCE DR. FENG ZHANG ON BEHALF OF THE RNA AND THE LAMDA LUNCH INTEREST GROUPS HERE AT NIH SO DR. ZHANG AS MANY OF YOU PROBABLY ALREADY KNOW HAS REVOLUTIONIZED SCIENCE IN ONE BUT TWO MAJOR WAYS BY CONTRIBUTING TO THE DEVELOPMENT OF OPTO GENETICS THE TECHNIQUE OF USING LIGHT TO CONTROL SPECIFIC NEURONS AND THE DEVELOPMENT OF CRISPER BASED TOOLS FOR GENOME EDITING AND AMAZINGLY YOUNG SO WHO KNOWS HOW MANY MORE IMPORTANT TOOLS HE IS GOING TO DEVELOP SO JUST A BRIEF BIOGRAPHY. DR. ZHANG WAS AN UNDER GRADUATE AT HARVARD WHERE HE WORKED WITH COLLEAGUES AND WENT TO STANFORD AND WORKED WITH DIZEROFF DEVELOPING OPTO GENETICS AND HE THEN MOVED BACK TO HARVARD WHERE HE WAS A HARVARD JUNIOR FELLOW AND WORKED WITH GEORGE CHURCH AS WELL. BACK AT HARVARD HE FIRST WORKED ON TALL OR TRANSCRIPTION ACTIVATOR LIKE PROTEINS AS TOOLS FOR GENONE AND EPIGENOME ENGINEERING AND QUICKLY REALIZED THE POTENTIAL OF THE CRISPR-CAS SYSTEMS FOUND IN BACTERIA AND ARCHAEA AS TOOLS FOR THE EDITING OF MAMALIA GENOMES AND DR. ZHANG AS APPOINTMENTS IN VARIOUS DEPARTMENTS AT MIT I WON'T NAME THEM ALL AS WELL AS AT THE BRODE INSTITUTE OF MIT AND HARVARD. AS MIGHT BE EXPECTED DR. ZHANG HAS BEEN AWARDED TOO MANY HONORS TO ENUMERATE HERE. I WILL JUST MENTION THE NIH DIRECTOR'S PIONEER AWARD, THE ALLEN T WATERMAN AWARD OF THE NATIONAL SCIENCE FOUNDATION AND THE GEDNER FOUNDATION AND TANG PRIZES THE LAST TWO WHICH HE SHARED WITH JENNIFER DOWNA AND MANWAL REGARDING THE DEVELOPMENT OF THE PRIPS CAS SYSTEMS FOR GENOME ENGINEERING. IN THE PAST SEVERAL YEARS THE ZHANG GROUP CONTRIBUTED MUCH TO OPTIMIZING CRISPR-CAS BASED GENOME EDITING AND THE UNDERSTANDING OF THE DIFFERENT CRISPR-CAS SYSTEMS AND DIFFERENT ORGANISMS AS WELL AS PHASED DEFENSES AGAINST THEM AND SORT OF LEARNING HOW TO LEVERAGE AND OPTIMIZE ALL OF THESE TOOLS. WHICH I THINK WILL BE THE TOPICS OF TODAY'S LECTURE. SO WITHOUT FURTHER ADIEU PLEASE JOIN ME IN WELCOME BACKING DR. FANNING ZHANG TO NIH. [APPLAUSE] WELL THANK YOU SO MUCH FOR THE KIND INTRODUCTION. IT'S TRULY A GREAT PLEASURE TO BE HERE AND I SPENT THE MORNING HERE INTERACTING WITH A NUMBER OF CLOSE COLLABORATORS AND COLLEAGUES AND FRIENDS AND IT'S BEEN TRULY A REALLY ENJOYABLE DAY HERE. BUT IT'S ALSO REALLY A TREMENDOUS HONOR TO BE HERE AT NIH AND TO SHARE WITH YOU SOME OF OUR WORK. NIH HAS BEEN THE KEY SUPPORTER FOR ALL OF THE WORK THAT WE HAVE BEEN DOING IN OUR LAB SO IT'S REALLY A TREMENDOUS OPPORTUNITY FOR US AND FOR ME TO BE ABLE TO TELL YOU ABOUT SOME OF THE THINGS THAT HAVE BEEN DONE UNDER YOUR SUPPORT. SO BY WAY OF THOUGHT GETTING STARTED WE HAVE BEEN WORKING ON DEVELOPI DEVELOPING MOLECULAR BIOLOGY AND THE STUDY OF BIOLOGICAL SYSTEMS AND ALSO TO ADDRESS IMPORTANT pPROBLEMS IN THE WORLD SOME OF THE SOLUTIONS COME FROM BIOLOGY. IF WE JUST LOOK AT YOU KNOW THE NEED FOR IMPROVED BIOLOGICAL TOOLS ALL THE WAY TO DEVELOPING MOLECULAR THERAPEUTICS AND TO DEVELOPING VISUALIZATION AND NEURO SCIENCE TECHNOLOGIES OR TO DEVELOP SUSTAINABLE ECOLOGICALLY FRIENDLY AND SUSTAINABLE RESOURCES, MANY OF THESE THINGS ARE MAJOR PROBLEMS FACING US AND THE SOLUTION TO SOME OF THEM LIE IN BIOLOGY. SO WHERE DOES IT COME FROM? SOME OF THE SOLUTIONS COME FROM THE ENORMOUS BIOLOGICAL DIVERSITY THAT SURROUND US. IF YOU LOOK AT THIS TREE OF LIFE, YOU CAN SEE THIS IS WHERE THEY ARE BUT IF YOU LOOK AT A BACTERIAL CELLS THEY REALLY REPRESENT SOME OF THE MOST AMAZING AND MOST ENORMOUS DIVERSITY OUT THERE. AND SO NEEDLESS TO SAY MANY OF THE ORGANISMS THAT LIVE OVER HERE HAVE OVER BILLIONS OF YEARS EVOLVED VERY NOVEL AND VERY POWERFUL MOLECULAR MECHANISMS TO ALLOW THEM TO SURVIVE IN DIFFERENT ECOLOGOLICAL AND HARNESSING SOME OF THEM WE CAN POTENTIALLY DEVELOP TRANSFORMATIVE BIOTECH GETS TO GIVE YOU A FEW EXAMPLES I HAVE COME OUT OF DIVERSITY IT FUELS THE DEVELOPMENT OF MOLECULAR BIOLOGY CAME FROM MICROBIAL ANTIVIRAL SYSTEMS AND VIRAL VECTORS THAT ARE PAVING THE WAY FOR GENE THERAPY CAME FROM NATURAL SOURCES ALL THE WAY TO FLUORESCENT PROTEINS AND OPTO GENETIC PROTEINS AND LOOK IN YOUR EVERYDAY HOUSEHOLD DETERGENT THERE ARE LIPAZES, CELUZAES AND ENZYMES THAT ARE HARVESTS FROM THE NATURAL SOURCES. AND SO INSPIRED BY ALL THIS WE CONTINUED TO SEE CAN WE EXPLORE THE NATURAL DIVERSITY MORE AND SOLVE IMPORTANT CHALLENGES THAT FACE OUR LIVES AND FACE HUMAN HEALTH. SO ONE OF THE PROBLEMS THAT WE FOCUS ON IS TRYING TO SEE CAN WE DEVELOP NEW SOLUTIONS TO BE ABLE TO ADDRESS THE EVER EXPANDING LIST OF GENETIC DISEASES. SEEMS THE COMPLETION OF THE HUMAN GENOME WE KNOW OVER 6,000 DIFFERENT GENETIC DISEASES CAUSED BY GENETIC MUTATIONS AND IF WE CAN GO IN AND TRY TO REVERSE THOSE MUTATIONS WE CAN POTENTIALLY DEVELOP TREATMENTS FOR THESE DISEASES, MANY OF THEM WE DON'T HAVE ANY TREATMENT FOR. SO THE SOLUTION TO THIS LIES IN AN AREA OF TECHNOLOGY CALLED GENOME EDITING AND THIS IS REALLY AN AREA THAT HAS BEEN PAVED BY GENE AND JAESEAN MORE THAN A DECADE ALMOST TWO DECADES AGO THEY REPORTED THE OBSERVATION THAT IF YOU CAN INDUCE THE NA DOUBLE STRAIN BREAKS IN SPECIFIC SITES IN THE GENOME THE BREAKS ARE REACTED BY THE CELL AS DNA DAMAGE AND THEN THEY WILL RECRUIT REPAIR MACHINERYS BELONGING TO ONE OF TWO DIFFERENT REPAIR PATHWAYS WENT JOYING AND HOMOLOGY DIRECT REPAIR AND CAN ALLOW US TO VERY EFFICIENTLY MAKE PRECISE ALTERATIONS TO THE GENOME. SO THEN BASED ON THIS INITIAL OBSERVATION MANY GROUPS HAVE ATTEMPTED TO DEVELOP A DNAENDO NUCLEUSS THAT CAN BE PREPROMISED TO INTRODUCE DOUBLE STRING BREAKS IN THE GENOME AND THIS IS THE BASIC IDEA IF WE CAN SOMEHOW TAILOR THE RECOGNITION DOMAIN FOR A SPECIFIC DNA SEQUENCE SO THAT IT CAN RECOGNIZE ANY SEQUENCE WE WANT IN THE GENOME THEN THIS DNA BINDING DOMAIN CAN BE USED TO BRING A NUCLEUS ACTIVITY TO A SPECIFIC SITE AND MAKE A DOUBLE STRAND BREAK. SO YOU CAN THINK OF THIS DOUBLE STRAND BREAK AS A CURIOUSER IN MICROSOFT WORD AND WE LOOK AT THE WORD PROCESS FOR LETTERS IN THE GENOME WHEREVER YOU CAN PLACE IT THAT IS WHEREVER YOU CAN MAKE THE EDIT. AND SO SEVERAL TECHNOLOGIES HAVE BEEN DEVELOPED TO MAKE IT POSSIBLE TO CUSTOMIZE THE BINDING AND RECRUITMENT OF THE NUCLEUS. SOME OF THE EARLIER TECHNOLOGIES INCLUDE FINGER PROTEINS AND ALSO TAIL PROTEINS BOTH OF THESE ARE PROTEIN ARE PROTEIN BASED DNA BINDING DOMAINS SO THAT MEANS THESE PROTEINS USE AMINO TO RECOGNIZE DNA SEQUENCES. THE COMPLEXITY OF REENGINEERING EACH ONE OF THE DNA BINDING DOMAINS CAN BE PRETTY CHALLENGING AND FINGERS HAVE INDIVIDUAL DOMAINS THAT CAN RECOGNIZE THREE BASES OF DNA AT A TIME BUT IN ORDER TO CUSTOMIZE THE RECOGNITION SPECIFICITY IT CAN TAKE QUITE A BIT OF DIRECTED EVOLUTION AND ENGINEER EFFORT AND EVEN THEN IT'S NOT GUARANTEED THAT WE WOULD DERIVE A FINGER PROTEIN THAT HAS HIGH EFFICIENCY AND ALSO CAN BIND VERY PRECISELY. SO ONE STEP BEYOND THAT WAS A SECOND SYSTEM THAT I PARTICIPATED IN DEVELOPING AND THIS IS A SYSTEM CALLED TALE SO TALE ARE FROM PLANTS PLANT PATHOGENS AND THEY HAVE REPEAT UNITS THAT ARE 34 AMINO ACIDS LONG AND ARE THE INDIVIDUAL COLOR MODULES AND MORE OR LESS IDENTICAL TO EACH OTHER EXCEPT TWO AMINO ACIDS AND IT'S THESE TO AND DEPENDING ON THE COMPOSITION OF THESE TWO AMINO ACIDS THAT DOMAIN CAN RECOGNIZE THE DIFFERENT DNA BASE. BECAUSE THERE ARE ONLY 40DNA BASES THAT DRASTICALLY SIMPLIFIES THE SYSTEM AND MEANS WE ONLY NEED FOUR DIFFERENT MODULES AND YOU CAN LINK THEM TOGETHER IN ANY COMBINATION YOU WANT TO RECOGNIZE A NEW DNA SEQUENCE OF CHOICE. BUT THIS SYSTEM STILL HAS ITS DOWN SIDES WHICH IS THESE MODULES ARE VERY REPETITIVE AND IF YOU WERE TO USE STANDARD PCR BASED GENO ASSEMBLY IT'S CHALLENGING TO ASSEMBLE A DESIRED COMBINATION OF THESE REPEAT UNITS SO WE THOUGHT MAYBE THERE ARE EVEN EASIER SOLUTIONS TO THIS AND ONE OF THE SOLUTIONS LIES IN A DIFFERENT SYSTEM THAT IS ALSO INVOLVED IN BACTERIAL DEFENSE. AND SO THIS IS A SYSTEM CALLED CRISPR-CAS, CRISPR-CAS STANDS FOR CLUSTER REGULARLY INTRO SPACE REPEAT AND THIS ACRONYM BASICALLY DESCRIBES THE NATURE OF THE REPEAT SEQUENCES THAT ARE THE SIGNATURE MOTIF OF A SINCE CRISPR-CAS SYSTEM AND THEY HAVE REPEAT ARRAYS WHERE THESE 30-36 TIED LONG ELEMENTS ARE FIXED BY NONREPETITIVE SEQUENCES CALLED SPACERS AND THESE SPACERS ARE NUCLE TIED SEQUENCES ACQUIRED FROM VIRUSS THAT INVADED THE CELL PREVIOUSLY SO EACH TIME THAT VIRUS COMES IN THESE PREVIOUSLY ACQUIRED NUCLEIC ACID SEQUENCES ACT AS MEMORY TO WORK TOGETHER WITH THESE PROTEIN BELONGING TO THE CRISPR-CAS SYSTEM TO BE ABLE TO MOUNT A RECOGNITION AND IMMUNITY RESPONSE AGAINST THE INVADING VIRUS. AND SO IF A VIRUS INJECTS ITS OWN DNA IN THE CRISPR-CAS RAY GETS EXPRESSED AS RNA AND PROCESSED DOWN INTO INDIVIDUAL RNA GUIDES, ONE SPACER PER GUIDE AND THAT CAN GET LOADED ON TO THE CAS PROTEINS TO FORM A COMPLEX THAT FACILITATES RECOGNITION OF THE RNA GUIDE WITH THE TARGET OF VIRAL DNA. AND IF THERE IS RECOGNITION THE ENZYME WILL GET ACTIVATED AND IT WILL CLEAVE AND FORM A DOUBLE STRAIN BREAK IN THE VIRUS DNA DOUBLE STRAIN BREAK WITH THE VIRUS INFECTION ACTIVATES THE VIRUS BUT WE THOUGHT MAYBE WE CAN TRANSPORT A SYSTEM INTO MAMALIA SALES WITH DOUBLE STRAND BREAK IN SPECIFIC SITES WHERE WE WANT TO PLACE THE GENOME EDITING CURIOUSER. SO I LEARNED ABOUT THE CRISPR-CAS SYSTEM WHEN I FIRST READ ONE OF THE PAPERS PUBLISHED BY SYLVIA WHO IS THE CANADIAN MICROBIOLOGIST SO BACK IN 2010 HE HAD REPORTED THAT IF THERE IS A SPECIFIC SEQUENCE IN THE CRISPR-CAS ARRAY AND IT MATCHES A SEQUENCE ON THE VIRUS THE PHAGE-DNA THE ENZYME WILL BE ON THE END OF THE RECOGNITION SITE AND THIS IMPLIES THIS IS RNA GUIDED ENDO NUCLEUS SYSTEM AND IF WE CAN HARNESS THIS SYSTEM AND MAKE IT WORK WITH CELLS WE CAN HAVE A MUCH EASIER TO REPROGRAM SYSTEM FOR ACHIEVING A GENOME EDITING SO WORKING WITH A NUMBER OF STUDENTS INCLUDING LOW AND ANN IN THE LAB WE DEVELOPED THE SYSTEM TO BE INTRODUCED CAS 9 AS WELL AS CRISPR-CAS RNA AND TRACER RNA INTO MAMALIA CELLS PREPROGRAMMING THE CRISPR-CAS RNA TO RECOGNIZE A SPECIFIC SEQUENCE ON GENOME THEY WANT TO EDIT SO IT WOULD GO WITH THE DNA AND CAS 9 CAN FACILITATE A BREAK AND STIMULATE THE REPAIRS IN THE CELL TO ALLOW GENOME EDITING. AND SO SINCE THEN WE HAVE ALSO WORKED ON A NUMBER OF OTHER MEMBERS AND WORKED ON TRYING TO BETTER UNDERSTAND THE PERIMETERS THAT GOVERN THE SPECIFICITY AND ALSO THE EFFICACYS OF THE SYSTEM AND THEN WORKING ALSO TO DEVELOP THE COMPUTATION ALGORITHMS WHICH IS SOMETHING THAT BEN HOLMES LED SO THAT REALLY UNDER LIES SOME OF OUR EARLIER WORK ON DEVELOPING THE CRISPR-CAS CAS 9 SYSTEM BUT TO SUMMARIZE THAT I WANT TO SHOW YOU A MOVIE HOW THE SYSTEM GENERALLY IS THOUGHT TO WORK. SO HERE YOU HAVE THE CAS 9 ENZYME WHICH HAS BEEN PRELOADED WITH RNA GUIDE PROGRAMMED TO RECOGNIZE A SPECIFIC SITE IN THE GENOME INTRODUCING THIS INTO THE HUMAN CELL NUCLEUS THIS ENZYME WILL TRY TO SEE IF THE RNA GUIDE CAN BASE PAIR WITH THE TARGET DNA SITE AND IF RNA BASE PAIRS THEN THE ENZYME WILL GET ACTIVATED AND IT WILL MAKE TWO CUTS IN THE TWO STRANDS OF DNA AND FORM A DOUBLE STRANDED BREAK. NW TWO DIFFERENT REPAIR SCENARIOS CAN HAPPEN. WITHOUT A TEMPLATE THE TWO DNA ENDS WILL REJOIN THROUGH A PROCESS OF END JOINING AND THIS IS AN ERROR PRONE PROCESS TO INTRODUCE A SMALL MUTATION THAT CAN ACTIVATE A GENE OF INTEREST AND GOOD FOR KNOCKING GENES OUT. ALTERNATIVELY WE CAN ACTIVATE A SECOND REPAIR PROCESS CALLED HOMOLOGY DIRECTED REPAIR AND THIS IS DONE BY PROVIDING ANOTHER PIECE OF DNA TEMPLATE THAT CARRIES HOMOLOGY TO THE TWO BROKEN ENDS AND IN BETWEEN IS A NEW DNA SEQUENCE WE WANT TO INTRODUCE IN THE GENOME AND THROUGH HOMOLOGY DIRECTED REPAIR IT CAN GET INCORPORATED IN THE GENOME ALLOWING US TO MAKE A VERY PRECISE CHANGE WITHIN A SPECIFIC SITE IN THE DNA. SO THAT'S TO FACILITATE GENOME EDITING BUT IN ADDITION TO CLEAVING DNA USING THESE TWO DOMAINS IN THE CAS 9 PROTEIN WE CAN ALSO INACTIVATE THEM SO WE CAN TURN THIS INTO A GENERIC BINDING DOMAIN AND WHAT IS SHOWN ON THE BOTTOM HERE. THE DNA BINDING DOMAIN CAN BE USED TO RECRUIT SPECIFIC EFFECTIVE MODULES TO DIFFERENT SITES IN THE GENOME. YOU CAN RECRUIT A TRANSCRIPTION ACTIVATION TO TURN ONE ON AND YOU CAN ACTIVATE TO BE ABLE TO MODIFY A SINGLE DNA BASE TO CHANGE IT TO A URO CELL AND BRING GFP IF YOU WANT TO VISUALIZE THE CONFORMATION DYNAMICS FOR AN LOCUST IN THE GENOME AND THERE ARE DIFFERENT WAYS WHERE THE CAS 9RNA GUIDED SYSTEM CAN BE USED TO STUDY GENOME FUNCTION. SO I THOUGHT I WILL SHARE WITH YOU A FEW OF THE WORK WE HAVE BEEN DOING RECENTLY AND SO SOME OF THE THINGS THAT WE HAVE BEEN DOING TO IMPROVE THE CAS 9 TARGETING SPECIFICITY AND THEN HOW WE CAN APPLY IT TO ACHIEVE HIGH THROUGH PUT GENOME SCALE TO LOOK AT GENE FUNCTION AS WELL AS MODELING SPECIFIC GENE MUTATIONS TO UNDERSTAND THEIR CONTRIBUTION IN DISEASE AND FINALLY SOME OF THE WORK THAT WE HAVE BEEN DOING IN COLLABORATION WITH OUR DEAR FRIEND EUGENE HERE TO EXPLORE CRISPR-CAS DIVERSITY TO BE ABLE TO FURTHER EXPAND THE CRISPR-CAS GENOME EDITING TOOL BOX SO WE WILL START WITH THE FIRST ONE. SO WE KNOW FROM MANY STUDIES BOTH RS AS WELL AS MANY OTHER GROUPS THAT CAS 9 CAN SOMETIMES INTRODUCE OFF TARGET ACTIVITY. SO BESIDES EDITING THE SITE THAT YOU WANT TO MODIFY IT CAN ALSO INTRODUCE OTHER UNWANTED MODIFICATIONS. SO WORKING WITH IAN AND THREE MEMBERS IN MY LAB WE LOOKED AT THE WAY THAT CAS 9 BINDS TO DNA AND THOUGHT MAYBE THERE ARE WAYS WE CAN REINTERNEENGINEER ONE OF THE FEATURES OF CAS 9 TO MAKE IT MUCH MORE SPECIFIC. THIS SKEMATIC SHOWS THE WAY THAT CAS 9 WOULD BIND TO DNA SO GRAY AND THE PURPLE AND BLUE OR GREEN ARE THE CAS 9 PROTEIN AND THE PURPLE AND THE GREEN SIGNIFY THE TWO CATALYTIC DOMAINS FOR CLEAVING DNA AND THE BOUND DNA SHOWN HERE IN RED AND THE GREEN IS THE RNA GUIDE. SO FOR THIS COMPLEX TO ACHIEVE RECOGNITION AND CLEAVAGE THE DNA WHICH IS NORMALLY IN DOUBLE STRANDED FORM NEEDS TO BE UNWOUND SO THAT ONE OF THE DNA STRANDS CAN BASE PAIR WITH THE RNA GUIDE. WHEN THAT HAPPENS, THE OTHER STRAND OF DNA NEEDS TO BE STABILIZED OTHERWISE IT WILL WANT TO REZIP AND REFORM AN HELIX AND REJECT THE RNA GUIDED AND PROTEIN SO FOR CLEAVAGE TO HAPPENING THIS BINDING EVENT NEEDS TO BE STRONG ENOUGH SO TO MAKE IT STRONG ENOUGH THIS DNA STRAND WHICH IS NEGATIVELY CHARGED NEEDS TO BE STABILIZED BY SOME POSITIVELY CHARGED PROTEIN AND SO WE LOOKED AT THE CRYSTAL STRUCTURE WHICH ALLOWS US TO EXAMINE THE SURFACE CHARGED DENSITY. THIS IS THE SAME CRYSTAL STRUCTURE FOR CAS 9 IT'S COLORED IN THE SAME WAY SO RATHER THAN LOOKING AT THE DOMAINS WE CAN RECOLOR IT TO LOOK AT THE SURFACE CHARGED DISTRIBUTION. AND SO HERE IS A DIFFERENT WAY OF COLORING IT. BLUE MEANS POSITIVELY CHARGED AND RED MEANS NEGATIVE CHARGE. AND SO YOU CAN SEE THIS GROUP HERE WHERE THAT IS PLACED DNA STRAND IS SUPPOSED TO FALL IS PRETTY BLUE SO WHAT THAT MEANS IS THAT THERE ARE A LOT OF POSITIVELY CHARGED AMINO ACIDS AND LIE SINES AND SO WE THOUGHT MAYBE THERE IS TOO MUCH POSITIVE CHARGE SO EVEN WHEN THE RNA IS NOT PERFECTLY MATCHED WITH THE DNA THIS STRAND IS SUFFICIENTLY STABILIZED THROUGH ACCESS POSITIVE CHARGE THAT THERE IS VERY LITTLE WILLINGNESS FOR THIS DNA TO REZIP WITH THE OTHER STAND AND EJECT THE COMPLEX AND THAT IS HOW YOU END UP WITH OFF TARGET DNA CLEAVAGE EVEN. SO WHEN WE EXAMINED THIS AND IDENTIFIED A LARGE NUMBER OF LYZENE RESIDUE AND IF WE CONVERT THEM TO SOMETHING THAT IS NOT POSITIVELY CHARGED WE CAN WEAKEN REACTION AND MAKE IT POSSIBLE FOR CAS 9 TO BE MORE SPECIFIC. AND SO WE SELECTED A NUMBER A LARGE NUMBER OF THESE MUTATIONS AND THROUGH TESTING WE FOUND THAT SOME MUTATIONS WERE ABLE TO MAKE CAS 9 SIGNIFICANTLY MORE SPECIFIC. SO HERE THIS IS THE WILD TYPE CAS 9 AND WE SELECTED A GUIDE RNA THAT TARGETS VEGFA AND GRAY BAR WE SEE ON TARGET VEGFA SITE AND FROM PREVIOUS WORK WE KNEW THE GUIDE HAD TWO OFF TARGET SITES O T1 AND O T-2 SO WITH THE WILD TYPE CAS 9 BOTH TARGET SITES WERE SIGNIFICANTLY MODIFIED AND SEE ALMOST 10% IN THE MUTATION WHICH IS VERY VERY HIGH. NOW, AFTER SCREENING A LARGE NUMBER OF MUTANTS WE IDENTIFIED TWO DIFFERET COMBINATIONS OF MUTATIONS BOTH COMBINATIONS CARRY THREE SUBSTITUTIONS THAT CONVERT LYZINE TO ALONINE AND THIS 1.0 PRESERVED ON TARGET EDITING AND ABOLISHED THE OFF TARGET EDITING ACTIVITY AND ALSO THE SECOND COMBINATION ALSO PRESERVED THE ON TARGET EDITING AND THEN WAS ABLE TO REDUCE THE EDITING TO THE SAME LEVEL AS BACKGROUND NOISE LEVEL AND SO FROM THIS WE KNEW THAT THROUGH THIS TYPE OF RATIONALE DESIGN PROCESS WE CAN MAKE CAS 9 MORE SPECIFIC SO THAT AS WE DEVELOP CAS 9 TO A HUMAN THERAPEUTIC WE CAN MAKE THE SYSTEM SAFER THROUGH THESE KINDS OF ENGINEERING APPROACHES. SO LET'S SWITCH GEARS A LITTLE BIT AND LOOK AT HOW WE MAYBE ABLE TO APPLY THE SYSTEM TO STUDY GENE FUNCTION. SO ONE OF THE NICE THINGS ABOUT THE RNA GUIDED SYSTEM IS THE RNA GUIDE CAN BE VERY EASILY SYNTHESIZED, IN FACT, WE CAN PRINT DNA AT VERY LARGE SCALE FOR VERY VERY LOW COST SO THAT WE CAN PROGRAM CAS 9 SIMULTANEOUSLY TO EDIT MANY MANY DIFFERENT GENES. SO USING COMPUTATION WE DESIGNED RNA GUIDES TO TARGET EVERY GENE IN THE GENOME AND DESIGN MULTIPLE GUIDES SO THERE IS SOME LEVEL REDUNDANCY SO WE CAN ACCOUNT FOR A POTENTIAL OFF TARGET EDITING ACTIVITY. AND THEN USING ALOGORY SYNTHESIS WE CAN SPRING -- PRINT IT USING INK JET PRINTING AND SIMILAR TO WHAT PEOPLE USE FOR THE LIBRARY TO TARGET THE WHOLE GENOME. WE CAN CLONE THE CAS 9 GUIDES INTO VECTORS AND PRODUCE VIRUSES THAT CAN THEN BE PUT ON TO A POPULATION CELL AT A LOW MULTIPLICITY INFECTION. THE REASON WE DO LOW MULTIPLICITY IS BECAUSE WE WANT TO INTRODUCE ONE VIRUS PER CELL AND NOT HAVE MORE THAN THAT, MORE THAN THAT WILL MAKE IT VERY DIFFICULT TO DECONVULVE TO FIGURE OUT WHAT IS THE MUTATION DOING THE INTERESTING PRESERVATION. WITH THIS POPULATION OF CELLS WE HAVE A BROAD ARRAY OF GENOTYPES EACH CELL RECEIVES A SINGLE GUIDE SO OVER A LARGE POPULATION CELLS WE NOW HAVE EVERY SINGLE GENE PERTURBED ONE GENE PER CELL WITH THE POPULATION YOU CAN APPLY A SELECTION ON THE CELL AND YOU CAN LOOK AT AND ASK QUESTIONS LIKE WHAT ARE GENES THAT WHEN YOU KNOCK OUT WILL RENDER THE CELL RESISTANT TO A CERTAIN VIRUS INFECTION OR WHAT ARE LOSS OF FUNCTION MUTATIONS THAT CONFER METASTATIC PROPERTIES FOR CANCER CELLS AND SO FORTH AND SO WE CAN LOOK AT THE RESULT OF THIS SELECTION BY SEQUENCING THE GUISE THAT ARE REMAINING IN THE SURVIVING CELLS AND BY MAPPING THEM AGAINST DIFFERENT GENES IN THE GENOME WE CAN IDENTIFY A LIST OF CANDIDATES THAT PARTICIPATE IN A SPECIFIC ROLE IN THE BIOLOGICAL FUNCTION. SO WE SHOW THAT YOU CAN DO THIS FOR BOTH LOSS OF FUNCTION SCREENS USING THE CAS 9 NUCLEUS SYSTEM TO INTRODUCE THEM AND YOU CAN ALSO PERFORM FUNCTION SCREENS WHERE USING CAS 9 TO RECRUIT A TRANSCRIPTION ACTIVATION DOMAIN YOU CAN TURN DIFFERENT GENES ON AND ASK THE INVERSE QUESTION OF WHAT ARE THE GENES THAT YOU NEED IN ORDER TO MEDIATE A SPECIFIC BIOLOGICAL PROCESS. SO THIS IS ALL REALLY NICE FOR STUDYING PROTEIN CODING GENES BUT WHAT ABOUT THE REST OF THE GENOME? AFTER ALL THE MAJORITY OF THE GENOME SEQUENCE DON'T CODE PROTEINS AND ASKED THE QUESTION CAN WE DEVELOP THIS AS A WAY TO STUDY THE FUNCTION NONCODING FUNCTIONS IN THE GENOME AND TWO OF MY FORMER LAB MEMBERS BOTH ARE NOW INDEPENDENT DEVELOPED A WAY TO BE ABLE TO DEVELOP RNA GUISE THAT TARGET PROMOTER OR THREE PRONG UNTRANSCRIBED TO INTRODUCE THEM IN A SATURATION WAY SO WE CAN ASK WHAT ARE THE NON-GENOME REGIONS FOR THE IMPORTANCE OF GENE REGULATION SO THE WAY JAESEAN AND NEVILLE DID IT IS TARGET THE POSSIBLE TARGET SITE FOR GENOME INTEREST AND USING THE SAME LIBRARY GENERATION METHOD WE CLONE THEM IN THE VIRUS LIBRARIES. SO THEN YOU CAN TAKE THE LIBRARY OF CELLS THAT HAVE DIFFERENT PRESERVATIONS ALONG THE NON-CODED REGION AND THEN APPLY A SELECTION AND IN THIS CASE WE LOOKED AT WHAT ARE GENOME ELEMENTS THAT PRETERB A SPECIFIC GENE AND DRIVE RESISTANCE IN THE PRESENCE OF A CANCER THERAPEUTIC. SO THIS IS THE GENOME WE TARGETED CALLED CALL THREE AND IT'S A GENE WE KNEW FROM A PREVIOUS SCREEN IF THE GENE IS INACTIVATED THEN A CELL WILL BECOME RESISTANT TO THE SELECTION AND WE DESIGNED GUISE THAT TILE ALONG THE REGION AND EACH ONE OF THE BLUE BARS YOU SEE HERE IS A TARGET SITE IN THAT GENOME REGION. AND SO BY PERFORMING THE SCREEN WE LOOKED AT THE SURVIVNG CELLS AND WE SAW THAT THERE ARE SPECIFIC GUISE ABOUT 7.2% OF THEM THAT BECAME INRUSHEINRUSH
ENRICHED AF
TER SELECTION AND WE ZOOM IN A LITTLE MORE AND SEE WHERE THE 7.2% OF GUISE. THIS IS THE GUIDE FREQUENCY PLOTTED ALONG THE LENS OF THE FIVE PRIME AND THREE PRIME REGION SO YOU CAN SEE THAT THERE ARE SPECIFIC AREAS WHERE CONCENTRATION OF THE GUIDES ARE OBSERVED, THAT ARE SURVIVING, THAT ARE ALLOWING THE CELL TO SURVIVE IN THE PRESENCE OF THE DRUG SELECTION. SO IF YOU ZOOM IN TO SOME OF THESE PEAK REGIONS YOU SEE THAT MANY OF THESE PEAKS REPRESENT TARGET SITES THAT BEAR MOTIFS TO KNOWN DNA BINDING SITES SO FOR EXAMPLE IF YOU LOOK AT THIS ONE, THIS ALSO OVERLAPS WITH THE BASICALLY BINDING SITE FOR THE YY1 TRANSCRIPTION FACTOR AND IF YOU LOOK AT A SPECIFIC DNA SEQUENCE YOU CAN SEE THEY CARRY A PARTICULAR MOTIVE AND IF YOU GO THROUGH ALL THE OTHER SITES YOU SEE MANY OF THEM REPRESENT KNOWN BINDING AND SOME POINT TO NOVEL DNA BINDING SITES AND USING THIS METHODOLOGY WE CAN BOTH STUDY PROTEIN CODING GENES AS WELL AS START TO MAP WITH VERY HIGH RESOLUTION SORT OF REGULATORY ELEMENTS IN A NONCODING GENOME. SO THIS IS TO SCREEN. THIS IS WHEN WE DON'T KNOW WHAT IS THE GENE THAT WE WANT TO STUDY AND WE WANT TO DISCOVER THE SPECIFIC GENETIC ELEMENT THAT DRIVES THIS TYPE OF BIOLOGICAL PROCESS BUT SOMETIMES WE ALREADY KNOW WHAT A GENE IS OR WE HAVE A LIST OF CANDIDATES AND WE WANT TO DIVE IN DEEPER TO UNDERSTAND THEIR FUNCTION. AND SO TO GIVE YOU A SENSE ABOUT HOW WE MIGHT DO THAT WE HAVE BEEN APPLYING THIS TO STUDY AUTISM SPECIALTY DISORDER. SO ABOUT SIX YEARS AGO THREE REALLY IMPORTANT PAPERS WERE PUBLISHED, WE ARE LOOKING AT THE DENOVO CODING MUTATIONS THAT ARE FOUND IN AUTISM SPECTRUM DISORDER PATIENTS AND SO THROUGH THESE THREE PAPERS THEY IDENTIFIED A NUMBER OF DIFFERENT MUTATIONS THAT ARE HIGHLY CORRELATED WITH AUTISM SPECTRUM DISORDER SO ONE NATURAL QUESTION I WHAT ARE THE CAUSAL ROLE OF THESE SPECIFIC GENES? SO ARE THEY JUST SIMPLY CORRELATED OR DOES IT ACTUALLY IMMEDIATE A CAUSAL RELATIONSHIP WITH AUTISM LIKE PHENO TYPE AND TAKING THE LIST OF CANDIDATE GENES WE USED GENOME EDITING TO TRY TO DEVELOP MODELS THAT ALLOW US TO STUDY THE FUNCTION OF THESE SPECIFIC GENES IN THE CONTEXT OF AUTISM. SO WE CONDUCTED TWO DIFFERENT LINES OF MODELING APPROACHES. THE FIRST ONE IS THE VITRO MODELING BASED ON STEM CELLS WHERE WE CAN BEGIN WITH A WELL CHARACTERIZED HEALTHY STEM CELL LINE AND USE THE GENOME SYSTEM THAT IS FOUND IN PATIENTS THE BENEFIT OF THIS IS WE CAN GENERATE A MUTANT CELL LINE THAT IS OTHERWISE ISO GENETIC TO THE HEALTHY UNMODIFIED STEM CELL LINES SO WE CAN CONCLUSIVELY SAY THE PHENO TYPE WE OBSERVED IS DUE TO THE GENE WE MODIFIED AND WITH MOUSE MODELS WE TAKE SINGLE CELL MOUSE EMBRYOS AND INTRODUCE THE SAME MUTATION WE TRIED TO MODEL IN HUMAN STEM CELLS SO N NEVILLE IS INDEPENDENT NOW AND CICI IS STILL IN THE LAB THEY INTRODUCED THE CAS 9 AND GUARD RNA INTO STEM CELLS AND THROUGH FACT SORTING THEY WERE ABLE TO SELECT OUT SPECIFIC CLONES THAT CARRY THE SPECIFIC MUTATION. AND SO THEN WE CAN DIFFERENTIATE THESE STEM CELLS INTO A NEURONS AND TRY TO SEE WHAT HAPPENS SO THESE ARE NEURONS THAT WERE DIFFERENTIATED BASED ON STEM CELLS AND PERFORMED PATCH CLIN EXPERIMENTS DOES THE PHYSIOLOGY CHANGE WITH THE CHANGE IN CHD8 AND LOOKING AT THE FIRING THRESHOLD WE INJECTED A STEADY CURRENT INTO THE CELLS AND WE MEASURED THE ACTION POTENTIALS THAT ARE EVOKED BY THIS INPUT OF ELECTRICAL ACTIVITY. WE FOUND THAT THE WILD TYPE UNMODIFIED CELLS WHICH IS THE CONTROL HAS SORT OF VERY NICE FIRING OF ACTION POTENTIAL TRAINS, EACH ONE OF THE SPIKES IS AN ACTION POTENTIAL. BUT THEN FOR NEURONS TO DERIVE FROM THE MUTANT CELLS, THE FIRING PATTERN IS MUCH MORE REPRESENTATIVE OF LESS MATURE NEURONS AND SO IF YOU LOOK AT THE SUMMARY STATISTICS YOU CAN SEE THAT THE NUMBER OF SPIKES THAT ARE FEARED BY THESE MUTANT CELLS ARE MUCH LOWER THAN THE NUMBER OF SPIKES FIRED BY THE WILD TYPE CELLS SO THIS SUGGESTS THAT MAYBE THE CELLS ARE POTENTIALLY LESS MATURE, MAYBE THEY ARE DEVELOPING OR DIFFERENTIATING A LITTLE SLOWER THAN A WILD TYPE CELLS. AND THEN WE DID A SECOND MEASUREMENT WHICH IS MEASURING SYNAPSE TRANSMISSION AND THIS IS SYNAPSE TRANSMISSION IN THE ESL NEURONS FOR CONTROL CELLS AND THESE ARE THE OTHER CELLS AND COMPARE THE CELLS WITH THE CONTROL CELLS YOU SEE BOTH THE AMPLITUDE SO HOW BIG THESE TRANSMISSIONS ARE AND ALSO THE FREQUENCY AT WHICH THE TRANSMISSION IS TRANSMITTED AS SPONTANEOUS ACTIVITY IN CULTURE CELLS IT'S BOTH LOWER COMPARED TO THE CONTROL. SO THESE ARE SUMMARY STATS SO YOU CAN SEE BOTH THE RATE AND ALSO THE AMPLITUDE ARE ALSO REDUCED. AND THAT IS CONSISTENT WITH THE SLOW DIFFERENTIATION OR SLOW MATURATION OF PHENOME TYPE AND IT LINKS IT WITH SPECIFIC GENOME TYPE OBSERVED IN THE HUMAN GENETIC STUDIES. SO IT'S HARD TO ASK -- IT'S HARD TO EXAMINE BEHAVIOR IN SPECIFIC NEURONS AND SO WE ALSO GENERATED MOUSE MODELS AND SO RANDY AND IAN TOOK MOUSE EMBRYOS IN THE DESIGN THE CRISPR-CAS REAGENT TO ALSO MODIFY CHDA GENE IN THESE MYSELF AND SIMILAR TO A HUMAN PATIENTS WE GENERATED HETEROZYGAS, CHD8 IN MYSELF TO STUDY WHAT HAPPENS ON THE BEHAVIORAL LEVEL. SO THERE ARE A NUMBER OF DIFFERENT HALLMARKS OF AUTISM. BEHAVIORAL HALLMARKS OF AUTISM SO WE RAN THE MYSELF THROUGH A NUMBER OF DIFFERENT BEHAVIORAL TESTS. THE FIRST ONE IS TO LOOK AT BOTH SOCIALABILITY AND ALSO SOCIAL NOVELTY SEEKING. THE WAY THIS WORKS IS AS FOLLOWS SO WE HAVE A THREE CHAMBER BOX, THERE ARE TWO SIDE CHAMBERS AND THE MIDDLE CHAMBER, WE PUT IN ONE OF THE SIDE CHAMBERS A MOUSE, A CONTAINER THAT HAS A MOUSE IN IT AND THE OTHER CHAMBER WE PUT IN AN EMPTY CONTAINER AND SO WE PUT A SUBJECT MOUSE IN THE MIDDLE CHAMBER AND THEN WE MEASURED THE AMOUNT OF TIME THAT THIS MOUSE INTERACTS WITH ONE OR THE OTHER JAR. AND SO IF THE MOUSE IS SOCIABLE ONE MEASURE USED TO MEASURE THAT IS TO SEE HOW MUCH TIME THIS MOUSE INTERACTS WITH THE JAR THAT CONTAINS ANOTHER MOUSE AND HOW LITTLE TIME IT SPENDS WITH EMPTY JAR. SO THIS IS TO MEASURE SOCIALABILITY. THE SECOND TEST TO MEASURE SOCIAL NOVELTY WHERE WE HAVE ONE JAR WITH A MOUSE THAT HAS BEEN PREVIOUSLY EXPOSED TO THE TEST SUBJECT AND THEN ON THE OTHER IN THE OTHER CHAMBER WE PUT A MOUSE THAT IS NOVEL SO IT HAS NEVER BEEN EXPOSED TO THE TEST SUBJECT AND THEN WE MEASURE THE AMOUNT OF TIME THAT THE TEST MOUSE SPENDS WITH THE FAMILIAR OR WITH THE DENOVO MOUSE AND THIS IS WHAT WE SAW ON BOTH METRICS ISOBARABILITY AND SEEKING AND THE HETEROZYGAS REDUCED SOCIALABILITY AS WELL AS SOCIAL NOVELTY SEEKING. SO THIS IS JUST ONE OF THE MANY BEHAVIORAL HALLMARKS SO THE SECOND ONE IS TO LOOK AT ANXIET AND SO HERE WHAT WE SAW IS THAT IF YOU PUT A MOUSE IN AN OPEN CHAMBER SO THIS IS JUST A SQUARE BOX AND THEN THERE IS A CAMERA ON TOP OF IT SO YOU CAN MONITOR THE PATH THAT A MOUSE WALKS IN THE BOX, AND HERE IS THE PLOT SHOWING THE PATTERN THAT THE MOUSE IS ABLE TO WALK AROUND, THE WILD TYPE MOUSE SPENDS TIME EVERY WHERE, IT ROAMS AROUND THE SENSOR AND THE SURROUND BASICALLY EVERYWHERE IN THE BOX BUT IF YOU LOOK AT CHD AND KNOCK OUT MOUSE IT ONLY STAYS IN THE PERIMETER OF THE BOX AND IT REALLY DOESN'T SPEND VERY MUCH TIME IN THE CENTER OF THE BOX. AND SO THIS IS A WAY TO MEASURE THE LEVEL OF ANXIETY THAT A MOUSE MIGHT BE EXHIBITING AND SO IF YOU QUANTIFY IN THE AMOUNT OF TIME SPENT IN SENSORY VERSUS THE SURROUND YOU CAN ALSO SEE CHD8 OF THE KNOCK OUT ANIMAL SPEND LESS TIME IN THE SENSOR THAN THE WILD TYPE MOUSE. THE THIRD HALLMARK WE EXAMINED IS THE ABILITY TO LEARN REPETITIVE TASK OR MOTOR LEARNING SO HERE THIS IS A BEHAVIORAL PARADIGM WHERE THERE IS A ROTATING ROD AND WE PUT THE TEST SUBJECT ON THE ROD AND WE MEASURE HOW MUCH TIME IT TAKES BEFORE THE MOUSE FALLS OFF OF THIS ROTATING ROD. THE LONGER IT STAYS THE BETTER IT'S ABLE TO PERFORM THIS MOTOR REPETITIVE LEARNING TASK AND SO THIS IS OVER MULTIPLE TRIALS AND YOU CAN SEE FOR BOTH THE WILD TYPE AND THE KNOCK OUT ANIMAL THE PERFORMANCE IMPROVES SO IT TAKES LONGER AND LONGER FOR ANIMAL TO FALL OFF BUT THE CHD8 KNOCK OUT ANIMAL IS ABLE TO LEARN SO MUCH FASTER SO THIS IS ANOTHER INDICATION THAT THERE IS SOME BEHAVIORAL DIFFERENCE IN THE CHD8 KNOCK OUT ANIMALS. SO AFTER SEEING ALL THESE BEHAVIORAL DIFFERENCES WE WANTED TO KNOW WHAT IS HAPPENING IN THE BRAIN OF THESE ANIMALS AND SO WE DISSECTED OUT TISSUE FROM DIFFERENT REGIONS OF THE CHD8 KNOCK OUT ANIMAL BRAIN AND COMPARED IT TO WILD TYPE AND OF ONE OF ONE OF THE BRAINS CALLED THE NUCLEUS WE SAW THE EXPRESSION OF NR2A AND NRTA, MMDA RECEPTORS IT WAS ELEVATED COMPARED TO THE WILD TYPE SO THIS PROMPTED US TO FOCUS IN MORE ON THE NUCLEUS ACCUMBENS TO SEE IF CHD8 MEDIATING ALL THESE OR CHD8 KNOCK OUT MEETING ALL THESE BEHAVIORAL PHENO TYPES THAT WE SAW AND THE TEST WE TURNED BACK TO THE CRISPR-CAS CAS 9 SYSTEM SO RATHER THAN MAKING A GLOBAL KNOCK OUT ANIMAL NOW WE BUILT VIRUSES TO BE ABLE TO DELIVER THE CAS 9 REAGENT INTO A SPECIFIC REGION SUCH AS THE NUCLEUS IN THE MOUSE BRAIN. WE HAD PREVIOUSLY BUILT AN ANIMAL THAT HAS A DEPENDENT CAS 9 SO WE THEN DESIGNED AB VIRUS THAT CARRIES BOTH AS WELL AS A GUIDE TARGETING CHD8 AND INJECTING THE VIRUS IN THE NUCLEUS WE WERE ABLE TO SELECTIVELY TRANS DUES THE NUCLEUS CELLS EXPRESSED AND ACTIVATE CAS 9 AND THEN USE THIS GUIDE TO KNOCK OUT CHD8 SELECTIVELY IN THE ACCUMBONS NEURONS AND WE CAN DO THIS QUITE PRECISELY. THIS IS INJECTION OF GHP SO THERE IS LOCALIZED EXPRESSION IN THE ACCUMBANCE AND WITH THE DORSAL YOU CAN GET EXPRESSION IN THE STRIATUM AND WE DID THE REGION SPECIFIC INJECTION TO SEE IS CHD8 A KNOCK OUT IN THIS ALONE ABLE TO MEDIATE SIMILAR BEHAVIORAL CHANGES. SO HERE WE FOUND THAT ON THE ROD TEST KNOCK OUT OF CHD8 IN THE INCUMBENTS ALSO INCREASED THE LEARNING PERFORMANCE WHEREAS KNOCK OUT CHD8 IN THE DORSAL STRIATUM AND THE REGION OR INJ EQUATION FOR THE GUIDE RFA THAT DOESN'T TARGET A MOUSE GENOME NEITHER OF THOSE IMPROVED THE LESHING PERFORMANCE ON THIS BEHAVIORAL TASK BUT WHEN WE TESTED THE KNOCK OUT ON SOCIAL OR ANXIETY TASK WE DID NOT SEE A SIGNIFICANT CHANGE IN BEHAVIOR AND THAT LIKELY SUGGESTS THAT OTHER BRAIN REGIONS ARE INVOLVED AND SO CHG8 IS PLAYING A ROLE IN OTHER BRAIN REGIONS TO REGULATE THOSE OTHER BEHAVIORS SO THIS IS SOMETHING THAT WE ARE CONTINUING TO DO TO TRY TO BETTER UNDERSTAND THESE GENES AND THEIR ROLES IN AUTISM SPECTRUM DISORDER AND HOPEFULLY BY SITTING SEVERAL DIFFERENT GENES WE CAN USE CONVERGING MECHANISMS DOWNSTREAM FROM THE DIFFERENT GENES THAT GOVERN THESE DIFFERENT AUTISM LIKE BEHAVIORS IN MYSELF. SO FINALLY JUST WANT TO SHARE WITH YOU SOME OF THE WORK ON EXPLORING CRISPR-CAS DIVERSITY WE ARE VERY EXCITED ABOUT AND PART OF THAT HAS TO DO WITH THE DIVERSITY OF CRISPR-CAS ASSISTANCE SO CRISPR-CAS CAS 9 IS THE MOST FAMOUS OF THE CRISPR-CAS SYSTEMS BUT MANY OTHER CRISPR-CAS SYSTEMS THAT EXISTS OUT THERE. AND SO WE HAVE BEEN VERY FORTUNATE TO COLLABORATE WITH EUGENE HERE AT NIH TO EXPLORE THE CRISPR-CAS DIVERSITY BUT LET ME TELL YOU WHAT THE DIFFERENT CLASSIFICATIONS ARE. BROADLY SPEAKING WE CAN DIVIDE THE CRISPR-CAS SYSTEM DOWN TO TWO CLASSES THERE IS CLASS ONE WHICH USE MULTIPLE PROTEINS TOGETHER TO FORM A LARGE MULTI SUB UNIT COMPLEX TO CARRY OUT THE RNA GUIDED RECOGNITION AND ALSO TO DOWNSTREAM DEFENSE FUNCTION. CLASS TWO WHICH IS THE CLASS THAT CAS 9 BELONGS TO IS MUCH SIMPLER AND USES A SINGLE PROTEIN WITH RNA GUIDE TO CARRY OUT THE RECOGNITION AND ALSO DEFENSE FUNCTION. AND SO WE THOUGHT MAYBE WE CAN EXPLORE THESE SINGLE COMPONENT CLASS TWO SYSTEMS AND SEE ARE THERE OTHER SYSTEMS OUT THERE THAT ARE POTENTIALLY QUITE POWERFUL AND WE CAN HARNESS THOSE TO DEVELOP A NEW BIO TECHNOLOGIES AND WORKING WITH EUGENE AND CARA AND KOSTYA COLLABORATOR IN RUSSIA WE USED COMPUTATION METHODS TO SEARCH THROUGH BACTERIAL SEQUENCES TO SEE ARE THERE OTHER CLASS TO CRISPR-CAS SYSTEMS. ONE VERSION OF THE SEARCH WORKS THIS WAY SO BY TAKING CAS ONE WHICH IS ONE OF THE MOST WELL CONSERVED CAS PROTEINS INVOLVING ACQUIRING IMMUNITY FOR THE CRISPR-CAS SYSTEMS. WE CAN IDENTIFY THAT CARRY THE CAS ONE PROTEIN. BECAUSE CRISPR-CAS SYSTEMS ARE ENCODED AND THERE IS A STRONG LIKELIHOOD THAT THE BY PROTEINS ARE ALSO RELATED TO CRISPR-CAS FUNCTION SO YOU CAN IDENTIFY THE OPEN READING NEARBY AND FOR EACH OPEN READING FRAME YOU CAN TRY TO ANNOTATE IT AS EITHER SOMETHING THAT IS SIMILAR TO WHAT WAS KNOWN BEFORE OR AS A COMPLETELY UNKNOWN SEQUENCE AND SO NATURALLY BECAUSE WE ARE INTERESTED IN FINDING UNKNOWN GENES FOCUS ON THE UNKNOWN SEQUENCES. AND THEN FOR EACH UNKNOWN PROTEIN THEN THERE ARE SEVERAL CRITERIA THAT WOULD BE APPLIED TO TRY TO SEE IS THERE A STRONG LIKELIHOOD THAT THIS CANDIDATE REPRESENTS A NOVEL CRISPR-CAS EFFECTED PROTEIN AND THE FIRST ONE IS WHETHER OR NOT THIS UNKNOWN PROTEIN REPRESENTS THE WHOLE FAMILY OF PROTEINS, IF THAT IS THE CASE THEN THAT REALLY SIGNIFIES THAT THIS IS EVOLUTION CONSERVED FAMILY OF PROTEINS. SECOND, MOST MEMBERS OF THE FAMILY COLOCATE WITH A CRISPR-CAS ARRAY AND IF THAT IS THE CASE THEN WE KNOW A IT'S EVOLUTION CONSERVED AND IT'S IMPORTANT BUT TWO IT'S ALSO A CONSERVED CRISPR-CAS PROTEIN BECAUSE IT'S OFTEN FOUND NEXT TO A CRISPR-CAS ARRAY SO BY APPLYING THIS TYPE OF METRIC WE ARE ABLE TO IDENTIFY A LARGE NUMBER OF DIFFERENT CANDIDATES AND THIS IS PROBABLY THE MOST OPPORTUNITY SINCE THIS OF THE CRISPR-CAS SYSTEM. SO THERE ARE TWO TYPES OF DNA TARGETING SYSTEM THERE IS THE TYPE TWO WHICH HAS CAS 9 AND TYPE 5 WHICH HAS CPF ONE OR WE NAMED TO CAS 12 AND ALSO OTHER RELATED FAMILY MEMBERS. AND THEN THERE IS THE TYPE 6 WHICH REPRESENT RNA GUIDED RNA TARGETED SYSTEMS AND THESE ARE CALLED CAS 13. THERE IS CAS 13A AND ALSO CAS 13B TWO DIFFERENT TYPES OF TARGETING SYSTEMS. SO WE HAVE BEEN STUDYING VARIOUS MEMBERS OF THE SYSTEMS AND WANT TO TELL YOU ABOUT SOME OF THE WORK WE HAVE BEEN DOING TO TRY TO UNDERSTAND WHAT CAS 13 MIGHT BE DOING. SO TWO OF MY GRADUATE STUDENTS OMAR AND ALSO JONATHAN FOCUS ON TRYING TO UNDERSTAND WHAT C2C2 MIGHT BE DOING, WORKING WITH THE NATURAL ORGANISM IS PRETTY CHALLENGING AND WORKING IT'S DIFFICULT WITH THE CULTURE AND ALSO IT'S DIFFICULT TO BE ABLE TO GENETICALLY MANIPULATE THEM SO INSTEAD OF WORKING WITH A NATIVE ORGANISM WE TOOK THE C2C2 LOCUST SO ALL OF THE CRISPR-CAS GENES AS WELL AS THE CRISPR-CAS ARRAY AND PUT IT INTO E. COLI SOMETHING THAT IS MUCH EASIER TO MANIPULATE THEN WE DID RNA SEQUENCING SO BY EXAMINING THE EXPRESSION OF RNA WE SAW THAT A CRISPR-CAS ARRAY IS NOT ONLY EXPRESSED BUT IT'S ALSO PROCESSED INTO THE INDIVIDUAL MATURE CRISPR-CAS RNA GUIDES SO THAT IS A VERY GOOD SIGN THAT SUGGESTS THAT MAYBE THIS IS ACTIVE CRISPR-CAS SYSTEM AT LEAST A CRISPR-CAS ARRAY IS PROCESSING INTO THE MATURE FORM. SO BASED ON COMPUTATION PREDICTION THERE ARE HEPN THAT ARE FOUND IN THE C2C2 PROTEINS AND THESE DOMAINS SUGGEST THIS MAY BE AN RNA TARGETING ENZYME RATHER THAN DNA TARGETING ENZYME AND SO TO TEST THE HYPOTHESIS WE DESIGNED A SCREEN TO SEE WHETHER OR NOT C2C2 IS ABLE TO HAVE IMMUNE DEFENSE AGAINST RNA VIRUS INFECTION. SO WE CHOSE AN RNA VIRUS CALLED MS2 THIS IS A SMALL VIRUS SO THE GENOME IS ONLY 3800 BASES LONG AND SO WE CAN DESIGN RNA GUIDES THAT TILE ALONG THE ENTIRE LENS OF THIS VIRUS'S GENOME AND SO THEN WE CAN SYNTHESIZE IT JUST LIKE THE WAY WE BUILD A CRISPR-CAS GUIDE IN OUR LIBRARY FOR SCREENING PURPOSES AND THEN WE CAN CLONE EACH ONE OF THE GUIDES INTO A VECTOR THAT CARRIES THE REST OF THE C2C2 SYSTEM SO EACH PLASMID CARRIES C2C2 SYSTEM AS WELL AS RNA OR C2C2 GUIDE THAT TARGETS THEM AS TO VIRUS SO THEN WE CAN TRANSFORM THIS PLASMA LIBRARY INTO E.COLI AND HAVE A LIBRARY OF E.COLI EACH CELL CARRIES A SINGLE TARGET SEQUENCE AGAINST MS2 AND THEN THE WHOLE LIBRARY TARGETS EVERY POSSIBLE POSITION WITHIN MS2 GENOME. SO IF C2C2 IS AN RNA GUIDED DEFENSE SYSTEM AGAINST RNA VIRUS THEN BY PUTTING THE MS2PHAGE ON THIS POPULATION SOME OF THE CELLS SHOULD SURVIVE AND SO IF WE SEE CELLS SURVIVE THEN WE CAN REASONABLY THINK THAT THERE IS RNA GUIDED, RNA INTERFERENCE AGAINST THIS MS2 VIRUS. AND THE FURTHERMORE IF WE LOOK AT THE SPACERS OR THE GUIDE SEQUENCES IN THE SURVIVING CELLS AND ANALYZE ANY SEQUENCE MOTIF THEN WE CAN USE THAT TO UNDERSTAND TARGETING REQUIREMENTS FOR THIS C2C2 BASED SYSTEM. SO WE DID THIS EXPERIMENT AND FORTUNATELY WE SAW THAT SOME CELLS DID SURVIVE AND SO THAT GOT US EXCITED AND WE THOUGHT MAYBE THIS IN DEED IS AN RNA GUIDED RNA DEFENSE SYSTEM. AND SO BY ANALYZING ALL THE GUIDE SEQUENCES IN THIS SURVIVING CELLS WE FOUND THAT RIGHT NEXT TO THE TARGET SITE WHICH IS CALLED PROTO SPACES AND CRISPR-CAS JARGON THERE IS A VERY SIMPLE MOTIVE WHICH IS A SINGLE BASE AND HAS TO BE A C OR A OR A U. AND AS LONG AS YOU HAVE THAT THEN THE C2C2 SYSTEM CAN SUCCESSFULLY MEDIATE DEFENSE AGAINST THIS MS2PHAGE. SO NOW WE THOUGHT TO SEE HOW WELL CAN WE USE THIS SYSTEM TO KNOCK DOWN A SPECIFIC RNA IN BACTERIAL CELLS. WE DESIGNED A DIFFERENT EXPERIMENT. NOW THERE ARE TWO PLASMAS ONE OF THEM CARRIES THE PROTEIN AND THE OTHER ONE CARRIES THE C2C2 SYSTEM AND A SPECIFIC RNA GUIDE DESIGNED BASED ON THE CA OR U MOTIVE TO TARGET THE RFP AND MRNA. AND SO BY TRANSFORMING E.COLI AND THEN MEASURING THE LEVEL OF THIS AND IF THERE IS DEGRADATION AND WE SHOULD SEE A DECREASE IN THE FLORESCENCE AND THAT IS WHAT WE SAW AND SO THIS IS THE CONTROL AND HERE IS A NEGATIVE CONTROL WHERE WE DESIGNED THE GUIDE THAT IS NOT FLANKED BY A C OR A OR A U SO THIS IS FLANKED BY A G AND THIS ONE HAS 100% FLUORESCENT AND FOR THE THREE GUIDES WITH THE RIGHT TARGET AND MOTIVE EACH ONE SIGNIFICANTLY REDUCED FLUORESCENT AND WERE NOT ABLE TO TARGET IT SUCCESSFULLY AND JUST TO MAKE SURE WE ARE NOT TARGETING THE RFP EXPRESSION PLASMA DNA WE THEN DESIGNED THREE GUIDES THAT TARGET THE TEMPLATE STRAND OF THE RFP GENE SO THIS IS TARGETING THE DNA AND HERE WE WERE NOT ABLE TO REDUCE THE RED FLOREESCENCE AND SHOWS A TARGETING SYSTEM AND THIS IS INTERESTING AND WE HAVE A SYSTEM WE CAN REPROGRAM WITH A SPECIFIC RNA TO TARGET OTHER RNA SPECIES BUT THERE IS SOMETHING MORE THAT IS INTERESTING ABOUT THIS SYSTEM. AND THAT IS IN ADDITION TO TARGETING THE RNA THAT IS BOUND BY THE RNA GUIDE AND THE C2C2 ENZYME UPON RECOGNITION THE C2C2 ENZYME CAN GET SWITCHED ON AND BECOME A NON-SPECIFIC PROMISCUOUS RNA IN SOLUTION AND SO WE IDENTIFIED THIS BY DESIGNING THIS EXPERIMENT SO HERE IS THE TARGET RNA BUT WE DON'T LABEL IT AND IF IT'S DEGRADED WE DON'T SEE IT WHEN WE RUN A GEL AND HERE IS THE RNA GUIDE. SO IN ADDITION TO THESE TWO RNA WE PUT IN A THIRD PIECE OF RNA THAT IS NOT RECOGNIZABLE BY THE RNA GUIDE BUT WE LABEL THIS RNA AND SO IF THIS RNA GETS CLEAVED THEN WE KNOW THAT WE CAN VISUALIZE IT BY LOOKING AT THIS GUIDE AND BY RUNNING THIS EXPERIMENT WE FOUND OUT THAT INDEED THIS RNA TRIGGERS THE CLEAVAGE OF THIS NON-SPECIFIC RNA. AND SO THIS IS THE GEL THAT SHOWS THAT SO HERE ARE TWO DIFFERENT TARGET RNA AND TO DIFFERENT VERSIONS OF THIS RNA AND WHENEVER IT'S DRESS ENAND TARGETED BY GUIDE RNA WE CAN SEE DEGRADATION OF THE RNA BUT THE YOU WITHDRAW THIS TARGET RNA SO IF THIS RNA IS NOT PRESENT SO IT'S ONLY C2C2 AND THIS CRISPR-CAS RNA THEN THERE IS NO DEGRADATION OF THE INPUT RNA AND WHAT IT SHOWED IS C2C2 REALLY HAS ADDITIONAL PROMISCUOUS ACTIVITY THAT ALLOWS IT TO CLEAVE RNA IN THE VICINITY BEYOND WHAT IS BOUND AT A TARGET SITE. SO THIS PARTICULAR FEATURE HAS POTENTIAL BIOTECH LOGICAL USES AS WELL AND SO USING THIS WE WERE ABLE TO DEVELOP A WAY TO DETECT INFECTIOUS AGENTS SUCH AS RNA BASED VIRUSES LIKE EBOLA AND ZIKA OR DETECT PATHOGENS IN THE DNA FORM. AND THE WAY THIS WORKS IS A PROCESS THAT WE CALLED SHERLOCK IT'S A TWO-STEP PROCESS. THE FIRST STEP OF THE PROCESS IS AMPLIFICATION STEF WITH ISO THERMO AND EASY TO PERFORM AND WHETHER IT'S INPUT DNA OR RNA WE CAN AMPLIFY IT AND USE T7 TRANSCRIPTION TO TURN IT TO RNA AND THAT IS THE MATERIAL WE THEN APPLY C2C2 AND THE DETECTION RNA TO TRY TO IDENTIFY. TO READOUT THE ACTIVITY WHAT WE DO IS IN ADDITION TO INCUBATING THIS RNA POOL WITH C2C2 AND GUIDE RNA WE HAVE A COMMERCIALLY AVAILABLE RNA REPORTER CALLED RNA ALERT AND THIS REPORTER HAS BOTH A QUINCER ATTACHED TO THE TWO END OF THE RNA SO IF C2C2 RECOGNIZES THE TAR TARGET AND CLEAVES IT IT WILL GET SWITCHED ON FOR OTHER IN THE SOLUTION AND CAN ALSO CLEAVE THE RNA ALERT REPORTER WHEN THAT HAPPENS THE QUINCEER ARE RELEASED FROM EACH OTHER AND YOU GET A FLUORESCENT SIGNAL. C2C2 IN THIS REACTION PROVIDES ADDITIONAL AMPLIFICATION BECAUSE EACH MOLECULE OF THE DETECTED RNA WILL SWITCH C2C2 INTO THIS NON-SPECIFIC PROMISCUOUS MODE AND THAT C2C2 CAN CLEAVE MANY THOUSANDS OF COPIES OF THE RNA REPORTER SO YOU GET AMPLIFICATION OF THE SIGNAL SO USING THIS YOU CAN READOUT THE PRESENCE OF THIS SPECIFIC RNA BY JUST LOOKING AT THE FLORESCENT EMISSION. AND SO WHAT WE FOUND IS THAT THIS SYSTEM CAN WORK PRETTY WELL. IN FACT, THE SHERLOCK SYSTEM WHICH IS THE C2C2 AND THE GUIDE RNA CAN BE FREEZE-DRIED ON TO A SHEET OF FILTER PAPER AND THAT IS REALLY NICE BECAUSE YOU CAN STORE IT VERY EASILY AND YOU CAN ALSO TRANSPORT VERY EASILY WITHOUT REQUIRING REFRIGERATION. AND THE WHOLE PROCESS WORKS VERY QUICKLY SO THIS IS YOU KNOW ABOUT 20 MINUTES OR SO TO BE ABLE TO PERFORM THE AMPLIFICATION AND THEN TO PERFORM DETECTION IT'S ANOTHER 20-30 MINUTES TO BE ABLE TO READOUT A SIGNAL SO IN LESS THAN AN HOUR YOU CAN PERFORM THIS DETECTION ACTUALLY. AND WITH OPTIMIZATION YOU WILL BE ABLE TO GET AS EVEN SHORTER PERIOD OF TIME. AND THE DETECTION WORKS VERY WELL SO WHEN WE DESIGN GUIDES TO DETECT ZIKA SEQUENCES WE CAN DETECT IT WITH VERY HIGH SENSITIVITY DOWN TO THE SORT OF TEN HOLDER RANGE SO THIS IS A LEVEL THAT WE NEED TO GET IN ORDER TO DETECT EARLY STAGES OF THESE VIRUS INFECTION. AND ALSO WITH OPTIMIZATION WE ALSO SHOW THAT YOU CAN GET IT DOWN TO EVEN A SINGLE DIGIT RANGE WHICH IS ABOUT ONE MOLECULE PER MICRO LITER OF CONCENTRATION. SO THIS IS FOR RNA DETECTION, THIS IS RNA SEQUENCES WITH ZIKA AND YOU CAN ALSO USE IT TO DETECT DNA SEQUENCES SO WE AMPLIFIED THE DNA AND USE T7 TRANSCRIPTION TO CONVERT DNA INTO RNA SO HERE THIS IS WITH SORT OF CLINICAL BACTERIAL CULTURE AND THE WAY IT RUNS THROUGH THE SAME PROCESS AND THEN FOR E.COLI WE CAN DETECT SPECIFICALLY USING E.COLI AND THE BACK IS LOW AND WE SIMPLY SWITCH TO PSEUDOMONAS WE CAN DETECT THAT AND THE BACKGROUND FOR OTHER BACTERIA SPECIES IS ALSO VERY LOW AND SO USING THIS KIND OF SYSTEM WE CAN VERY RAPIDLY AND ALSO SENSITIVELY DETECT SPECIFIC STRAINS AND YOU CAN CHANGE THE TEST TO DETECT ANYTHING YOU WANT JUST BY GIVING IT A NEW CRISPR-CAS RNA GUIDE AND THIS PROVIDES A VERY EFFICIENT AND EASY DIAGNOSTIC SYSTEM SO THIS REALLY HIGHLIGHTS HOW THERE COULD POTENTIALLY BE MANY MANY MORE POWERFUL BIO TECHNOLOGIES THAT ARE AWAITING DISCOVERY IN THE BACTERIAL DIVERSITY AND SO IF YOU JUST LOOK AT THE AMOUNT OF BACTERIA GENOME DATA THAT HAS BEEN ACCUMULATING OVER THE PAST TEN YEARS OR SO YOU CAN REALLY SEE THERE IS AN EX POTENTIAL GROWTH AND BETTER TO SEQUENCE AND ALSO ANALYZE THEM THERE IS VERY LIKELY ADDITIONAL TOOLS TO BE DISCOVERED AND I THINK IN THE COMING YEARS THERE WILL BE MANY MANY NEW IO TECHNOLOGY TO MAKE OUR LIVES IN HUMAN HEALTH MUCH, MUCH BETTER. SO THAT IS ALL I HAVE PREPARED FOR TODAY BUT I JUST WANT TO TAKE A MOMENT TO ACKNOWLEDGE MY TEAM AT MIT AND ALSO THE COLLABORATORS WE HAVE BEEN SO FORTUNATE TO WORK WITH AND MOST SPECIFICALLY EUGENE WHO HAS BEEN BOTH REALLY JUST A TREMENDOUS KNOWLEDGE BASE AND JUST REALLY GENIUS PERSON AND VERY LUCKY TO WORK WITH HIM AND THE TEAM TO STUDY THE CRISPR-CAS SYSTEM AND CHDA WE WERE ABLE TO WORK WITH ONE OF THE EXPERTS IN AUTISM MODELING AT MIT AND THEN FOR DEVELOPING THE DIAGNOSTICS WORKING WITH JIM COLLINS AND EXPERTS IN THE VIRUS AND BACTERIA INFECTION AND SO REALLY BEING ABLE TO WORK WITH A WORLD CLASS SET OF COLLABORATORS HAS MADE IT POSSIBLE FOR US TO DO THE WORK AND FINALLY JUST WANT TO ACKNOWLEDGE THE FUNDING AGENCIES BOTH AT NIH AS WELL AS SOME OF THE PRIVATE FOUNDATIONS AND INDIVIDUALS THAT MADE THE WORK POSSIBLE. THANK YOU VERY MUCH FOR YOUR INTEREST IN OUR WORK. [APPLAUSE] SO HAPPY TO ANSWER ANY QUESTIONS QUESTIONS. PLEASE USE A MICROPHONE IF YOU HAVE QUESTIONS. THANK YOU SO MUCH FOR SUCH A BRILLIANT TALK WE ENJOYED THAT VERY MUCH AND YOU MUST HAVE NOTICED THERE IS A NEW PUBLICATION AND THE NON-SPECIFIC MUTATION AND THAT INTRODUCED INTO A HOLDING SEQUENCE THAT IS THROUGH THE CRISPR-CAS PROCESS BUT THAT PAPER ALSO HAS CRITICS ABOUT SOME ERROR COULD YOU PROVIDE YOUR COMMENT AND WHAT IS YOUR OPINION ABOUT THAT, HOW TO AVOID THE OFF TARGET EFFECT OF THE CRISPR-CAS? >> SURE, YEAH, THAT IS A GOOD QUESTION AND IT'S AN IMPORTANT QUESTION. SO RECENTLY THERE IS A PAPER THAT REPORTED THERE MAY BE THOUSANDS OF SINGLE NUCLEO TIED INTRODUCED BY THE CAS 9 SYSTEM AND WE AND ALSO MANY OF THE LABS I HAVE WORKED WITH HAVE NOT OBSERVED THAT EFFECT AND IF YOU LOOK AT A PAPER CAREFULLY YOU MAY FIND THAT THE WAY THAT THEY MADE THE COMPARISON MAY NOT ALLOW FOR VERY CONCLUSIVE DETERMINATION OF THOSE SINGLE NUCLEI TIED AND THE MYSELF THAT WE INJECTED IT INTO IS A DIFFERENT GENETIC BACKGROUND THAN THE WILD MOUSE AND GIVEN THE STRAIN TO STRAIN CAN HAVE VARIATION IT'S POSSIBLE THAT THESE SMVS THAT THEY REPORTED TO BE CAUSED BY CAS 9 ARE ACTUALLY DUE TO A BACKGROUND DIFFERENCES WITH THE DIFFERENT STRAINS SO MORE WORK NEEDS TO BE DONE AND I THINK IT'S SOMETHING THAT HIGHLIGHTS HOW THAT SPECIFICITY IS REALLY AN IMPORTANT ISSUE AND SOMETHING WE NEED TO WORK ON. IN TERMS OF ACTUAL EXPERIMENT AND AVOIDING THIS I THINK THERE ARE A NUMBER OF THINGS THAT YOU CAN DO. THE FIRST IS TO COMPUTATIONALLY LOOK AT THE SEQUENCE YOU ARE DESIGNING AND MAKE SURE THAT IT IS AS DIFFERENT AS POSSIBLE FROM THE REST OF THE GENOME SO THAT YOU MINIMIZE ALL TARGET ACTIVITY. SECOND IS TO USE THESE ENHANCED VERDICTS OF CAS 9 WHERE WE INTRODUCE MUTATIONS THAT ALLOW THEM TO BECOME MUCH MORE SPECIFIC AND THEN FINALLY THE THIRD THING IS TO LIMIT THE EXPOSURE AND THE CONCENTRATION OF THE ENZYME THEY ARE INTRODUCING INTO CELLS SO RATHER THAN PUTTING A PLASMA THAT CAN EXPRESS THOUSANDS IF NOT TENS OF THOUSANDS OF ENZYME IN ITSELF FOR A LONG PERIOD OF TIME YOU CAN INTRODUCE THE PROTEIN RNA COMPLEX THE RNP INTO THOSE CELLS WHICH WILL BE MORE THE DOSE IS MUCH EASIER TO CONTROL AND ALSO IT WILL NOT LAST FOR AS LONG. SO IF YOU DO ALL THOSE THREE THINGS I THINK YOU CAN GET THE SPECIFICITY TO BE QUITE HIGH. >> AND SAME QUESTION VERY QUICKLY AND IS IT POSSIBLE TO DERIVE THE CRISPR-CAS SYSTEM DON'T REQUIRE A PAIRING SEQUENCE? >> THAT IS A REALLY GOOD QUESTION. SO THE PEM SEQUENCE IS A SMALL MOTIF THAT IS RIGHT NEXT TO THE TARGET SITE AND IT'S BEEN FOUND THROUGH A LOT OF EXPERIMENTATION THAT THE PEM IS WHAT CAS 9 USES TO LATCH ON DNA AND INITIATE UNWINDING AND SO IT'S POSSIBLE THAT MAYBE YOU CAN DESIGN A VERSION OF CAS 9 THAT HAS SUFFICIENT AFFINITY FOR GENERIC DNA SEQUENCE, NOT FOR A SPECIFIC BASE SEQUENCE TO ALLOW UNWINDING BUT EVEN IF THAT IS NOT EASY TO DO OR HARD TO GET AT IT'S POSSIBLE TO DESIGN CAS 9 TO REQUIRE SHORTER PEM SEQUENCES MAYBE A SINGLE NEUCLE TIED WITH FOUR VERSIONS OF CAS 9 SO YOU CAN COVER THE WHOLE SPACE. THANK YOU. >> I HAVE SORT OF A GENOME QUESTION SO YOU FOCUSED MOST OF YOUR TALK ON GENOME EDITING RIGHT AND THE ABILITY OF CAS 9 TO CUT DNA POSSIBLY TO INTRODUCE OR TO CORRECT MUTATIONS BUT IT SEEMS TO ME THAT THE PROMISE OF THE SYSTEM IS MUCH BROADER THAN THAT, RIGHT, I MEAN YOU CAN BASICALLY BRING TO A SPECIFIC REGION OF THE GENOME AND REGULATORY OR IMAGINARY SO IF YOU THINK IN TERMS OF IMPACT ON BIO MEDICAL RESEARCH ON NEW THERAPY MAYBE THE BROADER IMPACT WOULD BE REGULATING FOR EXAMPLE TRANSCRIPTION IN COMPLEX DISEASES, SO I WAS WONDERING WHAT WERE YOUR THOUGHTS ABOUT THE USE OF THOSE SYSTEMS BEYOND SIMPLY CORRECTING MUTATIONS? > I THINK YOU MAKE A VERY GOOD POINT. CERTAINLY BEING ABLE TO CUT DNA IS ONLY ONE OF MANY WAYS IN WHICH CAS 9 CAN BE ENGINEERED INTO A USEFUL TOOL AND SO THERE ARE FOLKS WHO ARE USING CAS 9 AS A NUCLEUS TO DEVELOP HUMAN THERAPEUTICS AND MANY NEW SYSTEMS THAT ARE BEING REPORTED TOO FOR EXAMPLE YOU CAN USE CAS 9 TO RECRUIT A TRANSCRIPTION pUSE IT TO RECRUIT CRAB DOMAINS TO REPRESS GENETICALLY A SPECIFIC LOCUS AND YOU CAN ALSO RECRUIT SITE TO BE ABLE TO CHANGE A SPECIFIC CITOZENE TO MAKE A C TWO 2 AND USE IT TO CAUSE VENTILATION AND GENE MODIFICATION SO ALL THESE DIFFERENT THINGS I THINK ARE VERY PROMISING AND I THINK THERE ARE MANY GROUPS THAT ARE WORKING ACTIVELY TO GET EACH ONE OF THOSE MODALITIES TO A HIGH ENOUGH LEVEL OF EFFICIENCY SO THAT WE CAN USE IT IN THESE KINDS OF APPLICATION CONTEXT INCLUDING FOR HUMAN THERAPEUTICS. FOR LONG-TERM EXPRESSION APPLICATIONS LIKE TO TURN A GENE ON OR TO TURN A GENE OFF, CHRONIC EXPRESSION OF A BACTERIAL PROTEIN IN HUMAN CELLS MAY POSE IMMUNO CHALLENGES SO THESE THINGS MAY -- THESE PROTEINS MAY GET PROCESSED INTO SMALL PEP SIDES AND PRESENT ON SMALL SURFACES AND CAUSE INFLAMMATION BUT THOSE ARE THINGS THAT ARE ALSO BEING ACTIVELY PURSUED AND STUDIED BY SEVERAL GROUPS SO I THINK IT'S VERY BRIGHT AT THE END OF THE TUNNEL THERE ARE MANY DIFFERENT APPLICATIONS AND I THINK EACH ONE OF THEM WILL CERTAINLY HAVE HUGE IMPACTS. >> HI, FOR YOUR CHD8 MYSELF HAVE YOU DONE HISTOLOGY TO LOOK AT CHANGES IN POPULATIONS WITHIN THE BRAIN THAT COULD EXPLAIN THE CHANGES IN BEHAVIOR THAT YOU SEE IN YOUR BEHAVIORAL STUDIES? >> YEAH, THAT IS A REALLY INTERESTING QUESTION SO WE HAVE DONE SOME WORK TO LOOK AT THAT SO FOR EXAMPLE WE HAVE FOUND THAT THE NUCLEI OF THE CHZ CELLS HAVE A DIFFERENT SIZE COMPARED TO WILD TYPE AND ALSO ANATOMICALLY THERE MAY BE DIFFERENCES WHICH WE STILL NEED TO ANALYZE SO CERTAINLY THERE ARE CHANGES TO THE CELLS. >> THANK YOU. >> THANK YOU. >> THANK YOU. >> SO PLEASE JOIN ME IN THANKING DR. FENG ZHANG ONE MORE TIME FOR A WONDERFUL SEMINAR. I WANT TO REMIND PEOPLE WHO ARE INTERESTED THERE IS A BRIEF RECEPTION NEXT DOOR IN THE NIH LIBRARY AND IT IS MY UNDERSTANDING THAT THIS IS THE LAST WALS LECTURE FOR THE 2016-17 SEASON SO LOOK FORWARD
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TO MORE GREAT TALKS NEXT FALL.
