>>  WELCOME TO A VERY SPECIAL 
LECTURE.  THE MARSHAL 
W NIRENBERG LECTURE IN THE
AUDITORIUM ON THE CAMPUS OF THE 
NATIONAL INSTITUTES OF HEALTH.
AND TO THE PEOPLE WHO ARE 
WATCHING ON THE VIDEO, WONDERFUL
TO SEE THE AUDITORIUM ABSOLUTELY
PACKED THIS AFTERNOON, WHICH I 
THINK REFLECTS THE IMPORTANCE OF
THE TOPIC AND THE SPEAKER THAT 
WE ARE FORTUNATE TO HAVE WITH US
NAMELY GEORGE CHURCH.
BEFORE I INTRODUCE GEORGE, LET 
ME JUST ALSO YOU SAY A WORD 
ABOUT MARSHAL NIRENBERG.
I AM ONE OF THOSE WHO HAD THE 
GOOD FORTUNE TO KNOW MARSHAL, 
AND OF COURSE, HE IS THE 
INDIVIDUAL WHO FIGURED OUT THE 
GENETIC CODE BACK IN THE EARLY 
60'S, BEGINNING WITH THAT 
REALIZATION THAT THAT POLY U 
CODES FOR FEN ALNINE AND GOING 
ON FROM THAT TO FILL OUT THE 
TABLE, SOMETHING THAT WE HAVE 
HAD MANY OCCASIONS TO REFLECT ON
SINCE MARSHAL IS ONE OF OUR OWN,
HAVING SPENT ALMOST HIS ENTIRE 
SCIENTIFIC CAREER HERE.
AND IF YOU HAVE NOT SEEN THE
DISPLAY ABOUT HIM AND HIS WORK 
THAT'S LOCATED OVER NEAR THE
LIPSETT AUDITORIUM, IN CASE YOU 
HAVE NOT WANDERED OVER THERE, GO
HAVE A LOOK.
IT'S PRETTY INTERESTING TO SEE 
SOME OF THE ORIGINAL MATERIALS 
AND EQUIPMENT THAT HE USED BACK 
THEN MORE THAN 50 YEARS AGO TO 
FIGURE OUT THIS FUNDAMENTAL 
INFORMATION, ABOUT HOW 
INFORMATION GETS TRANSLATED FROM
NUCLEIC ACIDS TO PROTEIN.
AND MARSHAL, AS YOU KNOW, DID A 
LOT OF THAT WORK IN THE AREA OF
MOLECULAR BIOLOGY AND IN THE 
SECOND HALF OF HIS CAREER, 
BECAME FASCINATEED WITH
NEUROBIOLOGY AND WAS ONE OF 
THOSE INDIVIDUALS WITH JUST 
BOUNDLESS CURIOSITY AND I 
CERTAINLY REMEMBER A NUMBER OF 
CONVERSATIONS WITH HIM AS HE WAS
NOT ONLY CHASING AFTER THE 
SECRETS OF NEUROBIOLOGY BUT 
BRINGING INTO THAT A WHOLE NEW 
SET OF IDEAS ABOUT CHEMISTRY AND
SMALL MOLECULE APPROACHES, WHICH
WERE A GREAT DEAL OF FUN TO
TALK ABOUT.
SO WE'VE HAD, SINCE THE LAST
FIVE YEARS, THIS LECTURE TO 
HONOR HIM IN PARTICULAR, WE HAVE
TRIED TO MAKE THIS AN 
OPPORTUNITY TO BRING IN SOME OF 
THE MOST HIGHLY RESPECTED 
FIGURES IN THE FIELDS OF 
MOLECULAR GENETICS AND TODAY WE 
HAVE ACTUALLY DONE EXTREMELY 
WELL IN THAT REGARD, BECAUSE WE 
HAVE A SPEAKER WHO IS GOING TO 
SPEAK ABOUT GENETIC CODES.
BUT ALSO ABOUT BRAIN CODES.
SO CAPTURING NEATLY BOTH PARTS 
OF MARSHAL'S CAREER IN THE WORK 
OF DR. CHURCH.
HE GOT HIS UNDERGRADUATE DEGREE 
AT DUKE.
HE WENT ON TO GET A PH.D AT 
HARVARD IN BIOCHEMISTRY AND 
MOLECULAR BIOLOGY.
AFTER A BRIEF STINT AT BIOGEN 
THEN AT UCSF, HE LANDED  BACK
AT HARVARD, WHERE HE HAS BEEN 
SINCE 1986 AND IS NOW A
PROFESSOR OF GENETICS, HEALTH 
SCIENCES AND TECHNOLOGY.
HE'S DIRECTOR OF THE HARVARD 
NHGRI CENTER OF EXCELLENCE IN 
GENOMIC SCIENCE, HAVING HAD THAT
ROLE NOW FOR ALL OF 13 YEARS.
THIS IS A MAJOR INVESTMENT THE 
GENOME INSTITUTE HAS MADE INTO 
DEVELOPMENT OF TECHNOLOGIES AND 
GEORGE HAS HAD A WONDERFUL TEAM 
THAT HAS BEEN WORKING ON THIS 
AND NO DOUBT WE'LL HEAR 
SOMETHING ABOUT THAT.
HE'S DIRECTOR OF THE PERSONAL 
GENOME PROJECT, THE FIRST EFFORT
TO DERIVE COMPLETE SEQUENCES ON 
INDIVIDUALS AND PUT ALL THAT 
INFORMATION TRULY IN THE PUBLIC 
DOMAIN, INCLUDING HIS /OEOWN.
HE'S A SENIOR ASSOCIATE OF THE 
BRODE INSTITUTE AND HE IS ALSO 
FOUNDING COFACULTY AND PLATFORM 
LEAD IN SYNTHETIC BIOLOGY FOR 
BIOLODGICALLY INSPIRED ENGINEER
ENGINEERING.
I AM HAPPY TO SAY I THINK I'VE 
KNOWN GEORGE FOR 30 YEARS AND 
THE WAY IN WHICH WE IN THOSE 
DAYS WERE TRYING TO DAYS FIGURE 
OUT HOW /TO SEQUENCE DNA AT THE 
PROPER SPEED THAT IT MIGHT 
ULTIMATELY LEAD TO THE WHOLE 
GENO
HUMAN GENOME AND THAT ULTIMATELY
SUCCEEDED AND IN NO SMALL PART 
BECAUSE OF THE CREATIVITY OF 
GEORGE AND OTHERS WHO CONTINUAL
CONTINUALLY INVENTED NEW WAYS TO
SPEED UP THE POSSIBILITY OF 
GETTING RAPID AND ACCURATE DNA 
SEQUENCE.
IN FACT, HE DEVELOPED ONE OF THE
FIRST DIRECT GENOMIC SEQUENCING 
METHODS.
HE'S ELECTED MEMBER OF THE 
NATIONAL ACADEMY OF SCIENCES AND
ALSO THE /TPHARNATIONAL ACADEMY
OF 
ENGINEERING.
AND HE WAS CITED AS ONE OF THE 
MAJOR CONTRIBUTORS, WHICH I 
SUSPECT HE MAY SAY SOMETHING 
ABOUT, IN SCIENCES BREAKTHROUGH 
OF THE YEAR, NAMELY CRISPER, 
WHERE GEORGE HAS, PLAYED A VERY 
SIGNIFICANT ROLE IN THE 
RESOLUTION THAT PROBABLY ALMOST 
EVERYBODY IN /THIS ROOM IS NOW 
USING IN THEIR OWN RESEARCH.
HE HAS NO LESS THAN 234070 PA
PAGEANTS.
SO WE ARE FORTUNATE INDEED TO 
HAVE DR. CHURCH WITH US.
PLEASE JOIN ME IN GIVING HIM A 
WARM NIH WELCOME.
/PHRA
[APPLAUSE]
-- PATENTS.
>>  THANK YOU VERY MUCH, FRANCIS
FRANCIS, AND EVERYBODY.
IT'S NICE TO SEE OLD FRIENDS 
HERE.
YOU'VE ALREADY SEEN ON THE PRE
PRESLIDES MY CONFLICT OF 
INTERESTS.
THAT'S WHAT THIS IS AS WELL.
LAUGHT[LAUGHTER] THERE IS
ACTUALLY A 
FULL WEB PAGE DEDICATED TO IT IF
YOU WANT /TO LOOK AT /THTHAT.
AND BE THETHERE WILL BE SOME
DRUGS 
MENTIONED, WHICH IS BY NO MEANS 
THE POINT OF THE TALK.
IT TURNS OUT THAT MARSHAL NEWER
NEWEREMBERG AND I HAVE HAD SOME 
PRETTY CLOSE RELATED PASSIONS.
I'VE BEEN WORKING ON GENETIC 
CODE SINCE I WAS A TEENAGER, 
ESSENTIALLY MY CAREER CAREER.
INITIALLY ON THE
CRIYSTALOGRAPHY,
WHICH IS TRANSRNA, WHICH IS THE 
THING THAT BREAKS THIS CODE.
WE'LL START ON GENETIC CODE AND 
HOW FAR IT'S COME AND WHERE IT'S
GOING.
AND THEN HE HAD MANY PAPERS ON 
NEWUROBLAST /OEPOMAS AND IN
PARTICULAR
ON TRANSCRIPTION FACTORS IN 
NEWUROBIOLOGY AND OUR APPROACH
IS
TRANSCRIPTION APPROACHES IN 
NEWUROBIOLOGY.
AND HE HAD AN INTEREST IN 
SCIENCE AND SOCIETY AND I'LL 
MENTION THAT BRIEFLY IN WRAPPING
UP.
SO THIS IS THE GENETIC CODE FOUR
YEARS AFTER HIS FIRST CODON WAS 
DISSECTED WHICH WAS U /SRAFU FOR
FEN ALGAE.
THERE IS A LOT /OF AMBIGUITY, 
ESPECIALLY THE LOEUCINE, TRIPTO 
FAN AND ARGNINE CODONS.
BUT MOST OF IT WAS DONE BY SIN 
THEY SIZING THESE NUKCLEOTIDES.
WHICH WAS A BIG DEAL IN 196 
/1K35* THEY MADE ESPECIALLY -- 
ESSENTIALLY ALL OF THEM.
AND POLYMERS OF THESE TRINUKE
TRINUCLEOTIDES.
THIS WAS -- AND BY THE WAY, PHIL
LITTER WAS ONE OF THE COSPIRIT
COSPIRITORS IN /THIS EFFORT AND 
HE WAS MY FIRST DEPARTMENT 
CHAIR.
SO WE'RE CONSTANTLY REMIND OF 
THE GENETIC CODE.
THIS IS WHERE WE ARE TODAY.
IN FACT, WE HAVE THE NUMBERS 
HERE ARE THE NUMBER OF INSTANCES
OF THESE -- EACH OF THESE CODON 
S IN A COMPLETE GENOME AND WE 
HAVE THESE FROM MANY DIFFERENT 
GENOMES.
THIS HAPPENS TO HAVE /* -- TO BE
ECO. COLI.
AND NOW THEY ARE THE ONES THAT 
WE'RE TARGETING FOR REMOVAL.
COINCIINCIDENTAL.
SO THEY ARE LOEUCINE, C /R-FPLR
ARGUE
ARGUENINE.
WE HAVE SYNTHESIZED THE 4 
MILLION BASE PAIRS, FOR WHICH 
THIS REPRESENTS FOR EACH TYPE OF
AMINO AJIHAD.
AND IN SO DOING, WE'VE HE WILL
ELIMINATED EITHER ONE OR 7 COD
CODONS GENOME-HON WIDE.
AND I'LL GIVE REASONS IN A 
MOMENT WHY WE DID THIS.
BUT THE THING THAT'S SIGNIFICANT
HERE IS WE NORMALLY READING 
ESSENTIALLY THE CODONS, BUT ALSO
NOW WRITING THEM.
AND WE CAN DO THIS RADICAL RE
RECODING FOR REASONS THAT WE'LL 
GET TO IN A MOMENT.
AND THE REASON THAT WE CAN DO 
THIS, THE REASON THAT WE CAN 
READ WHOLE GENOMES AND IN FACT 
/TPWAEBGT WRITE THEM ASSISTANT 
AT THE BILLION BASE PAIR SCALE 
IS THAT IT'S EX/PONENTIAL, AND
IT
WAS BREATH TAKING, EVEN BACK IN 
THE 1980S.
WE DIDN'T FULLY RECOGNIZE IT WAS
GOING AT /TTHAT RATE AND THEN 
AROUND 2003 AND 2004, IT CHANGED
SLOPE, WHICH TELLS YOU TO BE 
SUSPICIOUS OF THESE STRAIGHT 
LINES.
SO THIS IS FACTORS OF 10 ON THE 
Y /AB-AXIS.
AND NOW IT'S BEEN ABOUT A 3 
MILLION-FOLD IMPROVEMENT IN 
SEQUENCING AND ABOUT A BILLION-
BILLION-FOLD IMPROVEMENT IN SIN 
THEY SIS OF NUCLEOTIDES.
SO I'LL SPECULATE ON WHAT 
HAPPENED IN 2003 TO 2004 IN AN 
EXTREMELY IMMODEST WAY BY FOCUS
FOCUSING ON PAPERS THAT CAME 
FROM MY LAB IN 2003, 2004.
BUT IT ALSO -- AND THE KEY WAS 
GOING TO CHIPS, ESSENTIALLY 
PERFECTLY FLAT SURFACES, WAY 
LOUD US TO MINUIATURIZE, MULTI
MULTIPLEX AND START A NEW CODE 
THAT WAS MUCH FASTER.
AND THIS WAS -- WE WERE USING 
CHIPS TO SIN /TYNTHESIZE DNA TON
USE 
THEM THE WAY THEY WERE 
CONVENTIONALLY USE BUT TO STRIP 
OFF /THE DNA TO LOST ITS 
STRUCTURE SO THAT WE COULD A
ASSEMBLE LARGE GENOMES.
AND THERE IS AN INTERPLAY 
BETWEEN SEQUENCING AND
/STHEUPSYNTHESIS
SYNTHESIS.
IT'S CALLED SEQUENCING BY SIN
SYNTHESIS.
NOW IT IF WE GO FROM SIN
/TYNTHESIZE
SYNTHESIZING SHORT PIECES TO PUT
PEE TOGETHER LARGE GENOMES LIKE 
THE ONE THAT I BRIEFLY MENTIONED
MENTIONED, THIS IS OFTEN CALLED 
HE HAD -- EDITING.
I THINK THERE IS A VERSION OF 
THIS IS YOU WHICH IS CALLED 
WRITING THAT YOU EDIT SO MUCH 
THAT YOU HAVE TO SIN /TYNTHESIZE
HUGE
BLOCKS OF 50, 100 KILOBASES, A 
MEGOBASE.
BUT THE HE HEDITORS, THERE IS A
LOT 
OF OBSESSION RECENTLY AND 
FRANCIS ALLUDED TO THIS WITH 
CRISPER, WHICH IS IN THE UPPER 
RIGHT HERE.
BUT I WANT TO REMIND EVERYBODY 
THERE ARE AT LEAST NINE AND 
THERE PROBABLY WILL BE MORE AND 
EACH ONE /-OF THEM HAS BEEN DIS
DISPLACED OR FOUND ITS NICHE.
AND THEY HAVE THREE DIFFERENT 
WAYS OF SCANNING THE GENOME, 
THIS 6 BILLION BASE PAIRS IN THE
CASE OF HUMAN GENOME, TO FIND A 
UNIQUE PLACE TO MAKE AN /AEDIT O
OAIR CUT, AND THAT SCANNING HEAD
ESSENTIALLY, THE INFORMATION 
INTERFACE, IS EITHER DNA, RNA, O
OOR PROTEIN, EXAMPLES OF PROTEIN
SCANNERS.
NUCLEASES AND RNA AS THE CRISPER
CAST 9 AND DNA SCANNING ARE 
THINGS LIKE THE BETA RECOMBINASE
I'LL SHOW YOU IN A MOMENT.
NOW, THERE ARE PROBLEMS WITH 
CRISPER AND I WILL, EVEN THOUGH 
MY LAB WAS AMONG THE LABS THAT 
INVENTED IT, I'LL BE THE FIRST 
TO POINT OUT THE PROBLEMS OF ALL
OF OUR INVENTIONS.
AND THIS ONE HAS OFF-TARGET 
ISSUES AND ON TARGET ISSUES.
THE OFF-TARGET ISSUES HAVE BEEN 
MANY SOLUTIONS WE WON'T GO 
THROUGH THEM.
BUT ALL OF THEM CLAIM TO BE -- 
OR MANY OF THEM CLAIM TO BE SO 
LOW THAT YOU CAN CAN'T DETECT 
THE OFF-TARGET BUT THAT'S 
TYPICALLY BECAUSE THEY'VE USED 
THE TRICK THAT WAS USED IN THE 
VERY BEGINNING HERE 2013, WHICH 
IS COMPUTATIONAL, AVOIDING BEING
OFF BY MORE THAN 3.
SO YOU HAVE NO INSTANCES WHERE, 
WHERE YOU HAVE ONE OR TWO DIS
DISMATCHES.
THERE IS NOW ONE /WAWAY, IF YOU
ARE
INTERESTED OF GENERAL WAY OF 
GETTING SINGLE NUCLEOTIDE 
/KPWROEUL MORPHISM CHANGES 
SPECIFICITY AND THAT'S PUBLISHED
IN /THIS TOP-RATEED JOURNAL 
CALLED BIOARCHIVE, WHICH I EN
ENCOURAGE YOU ALL TO PUBLISH IN.
FROM MY GROUP.
SO THAT'S SINGLE NUCLEOTIDE-
NUCLEOTIDE-SPECIFIC CRISPER.
I CAN'T -- AND THEN THERE IS THE
ON TARGET PROBLEMS AND THIS 
COMES FROM ALL THE METHODS THAT 
USE DOUBLE STRAND BREAK.
TALONS AND CRISPER.
WHEN YOU MAKE A DOUBLE STRAND 
BREAK, IT'S A RACE BETWEEN THE 
CELL FIXING IT AND GETTING YOUR 
DONOR DNA TO FIX IT HOW YOU WANT
WANT.
AND THERE ARE TWO ALTERNATIVES 
WE'VE BEEN WORKING ON LONGER 
THAN CRISP ER AER AND THESE ARE
AS 
MANY THINGS.
THESE ARE HARVESTED FROM THE MI
MI/KROCROBIAL WORLD IN
PARTICULAR.
ECOLEA LAMD RECOMBINASE AND A
AVOID THE DOUBLE STRAND BREAK 
DILEMMA.
SO THE VARIOUS INT GRASSES 
REQUIRE PARING OF DONOR, EDITING
MOLECULE AND THE GENOME THAT YOU
ARE TRYING TO ENGINEER, AND THE 
BETA RECOMBINASE WORKS AT THE 
LAGGING STRAND TO PRETEND TO BE 
A FRAGMENT.
AND SO YOU ARE NOT MAKING A 
BREAK HERE.
YOU ARE JUST IN/KRORCORPORATE
ING ING A 
MISMATCH OR IT COULD BE A VERY 
LARGE MISMATCH.
SO WE'VE ACTUALLY USED THOSE TWO
NON-CRYSTAL METHODS.
SO I CALL THIS BEYOND CRISPER, 
TO DO THE LARGEST AND MOST 
RADICAL GENOME ENGINEER TO DATE,
WHICH IS CHANGING THE GENETIC 
CODE, BACK TO MARSHAL NEWEREM
NEWEREMBERG, OF ECO. COLI AND
HOPE
HOPEFULLY MANY OTHER ORGANISMS 
SOON.
AND REASONS ARE GIVEN HERE.
AND WE'VE CHANGED IT FIRST TO 
GET RID OF ONE CODON GENOME WIDE
WIDE.
SO THERE IS 64 TRIPLET CODONS 
AND ALL ORGANISMS SHARE ONE 
THING.
THEY DON'T ALL SHARE EXACTLY THE
SAME GENETIC CODE, ALTHOUGH IT'S
SIMILAR BUT THEY SHARE THAT THEY
USE ALL 64 CODONS SOMEHOW.
AND IN NATURE AND SYNTHETICALLY 
PART OF THIS WORK THERE WAS NONE
THAT USED 63 OR LESS.
SO NOW THERE IS ONE THAT USES 63
63, AND IT SHOWS ALL THE THINGS,
ALL THE GOALS THAT WE HAVE.
IT CAN USE NON-STANDARD AMINO 
ACIDS, NOT ORIGINAL TO THE SET 
THAT ALL ORGANISMS HAVE, 20, 21.
IT IS GENETICALLY ISOLATE SOD 
THIS COULD BE USED FOR BIO
BIOCONTAINMENT.
AND FINALLY HAS MULTIVIRUS RE
RESISTANCE.
I THINK THIS IS VERY INTERESTING
INTERESTING, BOTH PRACTICALLY 
AND PHILOSOPHYICALLY THAT YOU 
CAN MAKE AN ORGANISM RESIST
ANANT 
TO ALL VIRUSES IN THE WORLD THAT
YOU HAVE NEVER STUDIED BEFORE, 
BECAUSE THEY ALL EXPECT A GENT
GENETIC CODE TO BE PROVIDED BY 
THE HOST.
AND WE CAN CHANGE THAT IN A MORE
RADICALLY THE MORE VIRUSES.
BUT WE WERE SURPRISED THAT EVEN 
CHANGING ONE CODON WAS ENOUGH TO
MAKE IT RESIST /TAO*PT MOST 
CLASSES OF VIRUSES.
NOW, YOU MIGHT SAY WELL, WHY DO 
WE NEED NEW AMINO ACIDS 
/STPHEURGS MEAN, ISN'T 209 ADD
ADEQUATE?
QUICKLY JUSTIFIED -- JUSTIFY 
THAT, ONE OF THE FOUR GOALS.
THESE ARE SOME OF THE ONES THAT 
WE AND OTHERS HAVE USED TO 
ADVANTAGE.
YOU CAN PUT IN FLUORESCENT AMINO
ACIDS AND YOU CAN PUT IN MULTI
MULTIPLE FLUORESCENT AMINO ACIDS
ACIDS, WHICH IS MORE FASTER.
REDOX.
THERE IS A 10-FOLD /PROEUFPLT IN
REDOX IN SOME CASES.
RICK PLACE DISULFIDES WITH DISUL
DISULIN IDES.
YOU CAN GET OPTICAL WAVE LENGTH 
ISHE USOMERIZATION.
YOU CAN GET PEPTIC NUCLEOTIDE 
ACIDS BUT INEXPENSIVE ROOT.
AND FINALLY YOU GET CONJUGATES 
WHERE YOU CAN MAKE QUICK 
/KPHAEUPBLGSTRY COUPLEING AND 
THIS IS ALREADY IN USE IN THE 
PHARMACEUTICAL INDUSTRY TO MAKE 
PEGAL AATED HUMAN PROTEINS LIKE 
THIS IN CASE HUMAN GROWTH HORM 
ONE.
NOW, IN ORDER TO DO A WHOLEIEM, 
THE /STHEUSYNTHESIS IS NOT SO
BAD AND 
IT'S GETTING BETTER EXPONENTIAL
EXPONENTIALLY.
THE /RAREAL TRICK IS /-THE /*
DEBUG
DEBUGGING THE BIOLOGY.
SO WE WANTED TO DERISK THIS.
RATHER THAN BUILD A WHOLE GENOME
THAT WAS BROKEN IN 100 DIFFERENT
WAYS AND THEN TRYING TO FIGURE 
OUT WHAT WAS WRONG WITH IT.
WE WOULD BREAK IT UP INTO PIECES
AND DEBUG ALL THE PIECES.
AND INTERESTINGLY, WHEN WE DID 
THE ONE CODON, IT WAS EVERYTHING
ESSENTIALLY ALL 320 CHANGES 
WORKED.
SO THAT WAS A CASE WHERE WE WERE
PLEASANTLY SURPRISED WHEN WE DID
THE -- WHAT WE THOUGHT TO BE THE
TWO HARDEST CODONS, THEY WERE IN
INDEED HARD.
WE CHANGED -- THESE ARE THE UAG 
IS /THE MERIS CODON AND AG G
ANDA
ARE THE THEY COULDNSECOND AND
THIRD RAREST
 
ONES.
AND THERE WERE 13 EXAMPLES WHERE
THOSE WERE PROBLEMATIC AND WE 
HAD TO DEBUG IT AND EVENTUALLY 
CAME UP WITH WORK AROUND FORS 
ALL OF THEM.
THEN WE DID 13 CODONS FOR /AA
HUGE
FRACTION OF THE RIBOSOMAL GENES 
AND HERE WE ARE THE ONLY ONE 
WITH UNGOT YOU IN CHANGING THOSE
30 AND WE ENGINEERED AROUND THAT
THAT.
SO ALL OF THESE THINGS WE 
ENGINEERED AROUND AND THAT A
ALLOWED TO US MOVE AROUON TO THE
WHOLE GENOME, WHICH IS NOW 100% 
SIN /TYNTHESIZEED AND ASERVED IN
YEAST.
IT'S 86 PERCENT -- THESE NUMBERS
ARE A LITTLE OUT OF DATE BUT 
CLOSE ENOUGH.
86% IN EDOLEI AND VALID /A*ATE
AND 
IN/KRORCORPORATED THEM INTO THE 
GENOME.
AND THIS WAS A TEAM EFFORT, LED 
MOSTLY BY NELLY, WHO IS A POST 
DOCTORAL FELLOW IN THE LAB, AS 
WELL AS MICHAEL LANDEN.
NOW OUR GOAL IS NOT JUST TO HAVE
SOME GENOME THAT BARELY SAIS
ALIVE
ALIVE.
WE ACTUALLY WANT IT TO BE FASTER
THAN THE LABORATORY STRAINS.
AND HERE YOU CAN SEE IS OUR IN
INSPIRATION FOR THIS.
ON THE LEFT HA-HAND SIDE YOU
WILL 
FIND AN ORGANISM THAT IS DISTURB
DISTURBINGLY FASTER THAN ECO. 
COLI, WHICH IS FASTER THAN OTHER
ORGANISM ON THE PLANET.
THIS IS CALLED DAB RIO NETROGENS
AND IT'S WOKING -- BECOMING THE 
WORK HORSE FOR MOLECULAR BIOLOGY
THAT MAY RE/PHRAEPLACE ECOL.
COLI AND WE 
WILL BE SIN -- RECODING ITS 
GENOME OR MAKING OWE COLI HAVE 
THE PROPERTIES THAT WE WANT.
ECO. COLI.
SO TO THE SECOND TOPIC, HAVING 
TO DO WITH MARSHAL NUREMBERG, 
WAS BRAIN CODES.
AND I CAPITAL /AOEIZE THE WORD
BRAIN 
FOR SOME OF YOU THAT MAY KNOW.
A FEW OF MY COLLEAGUES AND I HAD
AN /OBSESSION WITH BRAIN
ACTIVITY
MAPPING AROUND 2011, WHICH 
MANAGED TO MAKE IT TO THE 
ATTENTION OF THE OS TP AND NIH 
AND OTHER VARIATI/SKPAOGSZ IT
BECAME A 
REAL THING, WHICH IS NOW CALLED 
BRAIN RESEARCH THROUGH ADVANCING
ONINNOVATIVE
NEWEUROTECHNOLOGIES.
SO THAT'S THE AKRCRONYM AND I 
REALLY LOVED IT WHEN IT WAS 
ANNOUNCED, BECAUSE IT INCLUDED 
THE WORDS ONINNOVATIVE AND 
TECHNOLOGIES, WHICH IS NOT 
SOMETHING YOU OFTEN GET FROM A 
TOP-DOWN PROJECT.
AND IT SHAPED UP HAVE /* TO BE A
VERY REMARKABLE PROJECT AND I'LL
TELL YOU A LITTLE BIT ABOUT OUR 
LITTLE PIECE OF IT.
SO MARSHAL NUREMBURG WORMKED ON 
KREBS AND SO FORTH AND I'LL TELL
YOU WHAT WE'RE DOING NOW AND IN 
THE NEAR FUTURE THAT 
SPECIFICALLY INVOLVES BUILDING 
BRAINS, NOT JUST ANALYZING THEM 
BUT BOTH.
SO IT'S THE WHOLE READING AND 
WRITING MANTRA THAT WE HAVE FOR 
THE GENETIC CODE AND THE GENOME.
WE CAN ALSO DO FOR THESE EP
EP/SKWREPIGENETIC CODE.
AND AS YOU WILL SEE IN A MOMENT,
WE TRIED VARIOUS WAYS OF DOING 
THE EP/SKWREPIGENETIC
REPROGRAMMING, 
INCLUDING USING CRISPER, BUT WE 
FOUND THAT IN DIRECT SIDE-BY-
SIDE-BY-SIDE COMPARISONS, WE 
COULD GET A LOT MORE MILEAGE 
RIGHT NOW USING TRANSCRIPTION 
FACTORS.
AND WE WERE SHOCKED TO FIND OUT 
THAT DESPITE THE FACT THAT WE'VE
GOT C /TK-FPDNA /STKWAOEFPBLTSS
FOR 
MANY HUMAN CNAS AND THERE WAS NO
COLLECTION OF HUMAN GENES OR 
HUMAN TRANSCRIPTION FACTORS THAT
WAS ADEQUATE, EVEN CLOSE TO /AD
ADEQUATE.
SO ALEX, AS /AA GRADUATE STUDENT
IN THE LAB, PUT TOGETHER 1576 
HUMAN TRANSCRIPTION FACTORS.
WE THINK THIS IS AT LEAST ONE 
PER GENE THAT IS CLASSIFIED EVEN
IN /AA BROAD CLASSIFICATION OF 
WHAT A TRANSCRIPTION FACTOR IS.
88 OF THEM HAVE ALTERNATIVE 
SPLICE FORMS AND THOSE ARE QUITE
USEFUL AND WE'LL PROBABLY KEEP 
EXPANDING THAT.
BUT THERE IS AT LEAST ONE 
TRANSCRIPTION FACTOR PER 
TRANSCRIPTION FACTOR GENE.  AND 
AND WE'VE HAD MANY APDATABA-- AP
APPLICATIONS.
EVEN THOUGH IT HASN'T BEEN 
PUBLISHED, WE HAVE EIGHT 
COLLABORATORS THAT FOUND OUT 
ABOUT IT THROUGH TALKS LIKE THIS
THIS.
AND HERE IS AN EXAMPLE OF WHERE 
WE SCREENED IT FOR LOSS OF PLURI
PLURIPOTENCY IN /AEA HUMAN STEM 
CELL LINE.
MORE ABOUT THAT IN A MOMENT.
AND WHAT YOU CAN FIND IS THAT 
ONE END /-OF THE SPECTRUM ARE 
THINGS THAT STABILIZE PLURIPOET
PLURIPOTENCY AND AT THE OTHER 
END /-OF THE SPECTRUM ARE
FACTORS 
THAT TAKE IT TO DIFFERENTIATE 
INTO SOMETHING.
AND IT IS AN INTERESTING OPEN 
QUESTION, BUT WE'VE USED THIS TO
FIND A WHOLE VARIETY OF NEW PATH
PATHWAYS, SOME OF WHICH ARE 
EXTRAORDINARILY EFFICIENT AND 
RAPID.
SO FOR EXAMPLE, WHEN SOME OF THE
PROTOCOLS WE WERE LOOKING AT FOR
SOME OF THE TISSUES REQUIRED -- 
THE PROTOCOLS REQUIRED 120 DAYS 
AND MAYBE HAVE SINGLE DIGIT 
EFFICIENCIES.
WHILE HERE WE HAVE NOW NUMEROUS 
TRANSCRIPTION FACTORS THAT WILL 
GIVE US GUST 98 -- GIVE US 98% 
YIELD OF NEWURONS AND
ESSENTIALLY
WE DON'T HAVE TO /REMOVE THE
STEM
CELLS THAT ARE COMPLETELY GONE 
AND THE 2% THAT AREN'T NEWURONS 
ARE NOT STEM CELLS EITHER.
BUT THE POINT IS IT'S VERY 
EFFICIENT AND SHORT PERIOD OF 
TIME.
AND HERE IS THE TWO METHODS THAT
I'VE ALLUDED TO /TTHAT WE'VE
TRIED
TRIED.
WE DEVELOPED ONE OF THE MOST 
EFFICIENT CRISPER BASE.
SO THIS IS DEAD CAST 9 WITH 
THREE DIFFERENT ACTIVATION DO
DOMAINS ATTACHED TO A C TERMINIS
TERMINIS.
AND THIS IS ABOUT 20,000 TIMES 
MORE ACTIVE -- CAN INCREASE GENE
EXPRESSION BY 20,000 TIMES, AND 
IT'S 100 TIMES MORE ACTIVE THAN 
THE FIRST CRISPER ACTIVATORS 
THAT WE AND OTHERS DEVELOPED.
ANYWAY, WHEN WE HOOKED THIS UP 
TO NEWUROJEGENIC, WE GET ABOUT
7% 
FIELD /* YIELD, BUT WE /- WHEN 
WE GO TO NEWUROOJEGENUINE
DIRECTLY 
TO TRANSCRIPTION FACTOR, WE GET 
98% YIELD.
AND I WANT DAY WE'RE ALREADY 
SEEING THIS HUGE TRANSFORMATION 
AND SORT OF THIS GREGARIOUS PILE
OF STEM CELLS, WHICH -- TO THESE
ELONG AATED BIPOLAR NEWEURONS,
WHERE
YOU HAVE EXACTLY TWO PROCESSES.
NOW, ONE OF THE CHALLENGES, WHEN
WE DEVELOPED THESE PROTOCOLS, 
WHERE WE GET A NEW DEVELOPMENTAL
FATE IS /TKW-- FIGURING OUT WHAT
IT 
CORNERS TO BUT DESPITE ALL THE 
TRANSCRIPT /O*EOMICS THAT'S
OCCURRED 
IN THE WORLD, WE STILL DON'T 
HAVE A LIST OF ALL THE 
TRANSCRIPT OHMS FOR ALL THE CELL
TYPES IN ALL THE STAGES OF 
DEVELOPMENT SO WE DON'T KNOW 
WHAT EXACTLY THIS CELL 
CORRESPONDS TO YET.
BUT WE AND OTHERS ARE WORKING ON
A CELL ATLAS FOR HUMAN AND MOUSE
THAT WILL COVER HOPEFULLY EVERY 
CELL STATE AND EVERY STAGE OF 
DEVELOPMENT.
IT /TKWRUFPBLTS -- MOST OF THE 
TRANSCRIPT OHMS HAVE BEEN DONE 
ON BLOCKS OF TISSUES THAT HAVE 
SPECIFIC CELL TYPES.
THAT'S THE CONVERSION.
IT'S VERY FAST AND EFFICIENT AND
WE'VE DONE IT FOR /AA LARGE
NUMBER
OF CELL TYPES.
NEWURONS, MYEIOCYTES,, AND HERE 
ARE SOME OF THE CASES WHERE ON 
THE TOP ARE THE GENES, THE 
TRANSCRIPTION FACTORS THAT WE'VE
USED AND DOWN BELOW ARE SOME OF 
THE ANTIBODIES THAT WE USED TO 
SHOW WHAT CELL TYPE IT IS OR TO 
DO FACT SORTING.
AND THEN WE FOLLOW UP WITH 
TRANSCRIPT /O*EOME ON THESE CELL
TYPES.
AND /THAOS WORK FROM ALEX, AGAIN
AGAIN, AND /AA POST DOC IN THE
LAB
LAB.
NOW THE NEXT STEP /UP FROM CELL 
TYPES, TISSUE TYPES, IS ORGANIS 
OR ORGANIOIDS, AS THEY ARE
MODEST
MODESTLY CALLED.
WE HAVEN'T GOTTEN PERFECT 
ORGANIORGANS
YET.
AND THERE IS PIONEERING WORK 
FROM LAN /KCASTER IN 2013, WHERE
THEY ACTUALLY USED THESE TWO /* 
TO STUDY MI/KCROSELCEPHALY GENE
GENT
GENETIC INFLUENCES THAT SHOW 
CAUSE AND EFFECT IN VARIATION IN
A WAY THAT'S VERY HARD TO DO IN 
MICE.
IT'S HARD TO GET BECAUSE OF THE 
AN /TOATOMICAL STRUCTURES ARE
NOT 
PRESENT.
BUT THEY ACTUALLY GOT GOOD AN 
TOMICAL STRUCTURES.
YOU CAN SEE THIS BEAUTIFUL 
LAYERED EFFECT.
BUT IF THEY GET MORE THAN HALF A
MILLIMETER, YOU START GETTING NE
NECROSIS BECAUSE THERE IS NO VAS
CLATOUR.
WE SET OUT TO SOLVE THE PROBLEM 
USING THE SAME KIND OF PLURIPOET
PLURIPOTENT STEM CELLS, HUMAN IP
IPS, THAT WE WERE USING BEFORE, 
BUT NOW USING THE TRANSCRIPTION 
FACTORS IN MULTIPLE COMBINATIONS
TO GET AT VAS /KHCULATURE.
NOW THE CHALLENGE WAS HERE IF 
YOU JUST TAKE NEWURONS OR NUR
OWN
OWNAL PRE/KUCURSORS IN
ENDO/THAOETHELIAL 
CELLS AND MIX THEM TOGETHER AND 
HOPE FOR THE BEST, THEY WILL 
SEPARATE LIKE OIL AND WATER.
THAT HAS BEEN OUR EXPERIENCE.
BUT IF YOU PUT IN INDUCED PLURI
PLURIPOTENT STEM CELLS THAT ARE 
UN/TKEDIFFERENTIATED, THEY LOVE
TO 
AGGREGATE AND IF YOU HAVE A MIX
MIXTURE WHERE THEY ARE 
PROGRAMMED IN ADVANCE TO DO 
DIFFERENT THINGS WHEN YOU ADD 
THE DEOXY CYCLIN INDUCEER, YOU 
WILL GET THE AGGREGATION AND GET
INTER/SPESPERSED SELF-ASSEMBLING
NOW
VASCULARIZED ORGANIOID S.
THE PAPER HAD ORGANIOIDED BUT
NOW
YOU HAVE THIS ORGANIZE -- 
ORGANIZATION WITH VAS
/KHCULATURE 
MIXD IN.
AND HERE THE CAPILLARIES HAVE 
BEEN HERE LABELED WITH ANTI-
ANTIBODIES.
/SREFRPLT CAD HEROIN AND ARE 
NICELY INTER/SPESPERSED WITH THE
VARIOUS NEWURONAL TYPES.
WE'VE ALREASO, IN ADDITION TO
/-THE 
CAP /HRILLARIES AND THE
NUEURONS, WE 
HAVE OTHER NON-NUR /O*EURONAL 
COMPONENTS.
THE NEXT TEP IS HOOKING THIS UP 
EITHER TO MI/KCROPUMPS SUCH AS
THE
ONES THAT ARE ALREADY COMMON IN 
OUR LAB AND IN JENNIFER LIEWIS'S
OR GETTING IT TO PUMP ITSELF 
WITH A CARDIAC MUSCLE THAT YOU 
WILL SEE IN /AA SUBSEQUENT
SLIDE.
NOW, JUST /AA SLIGHT DIGRESSION 
HERE.
ALL OF THE HUMAN
EP/SKWREPIGENETICS 
THAT WE'VE SHOWN SO FAR IS DUE 
TO THE PERSONAL GENOME PROJECT 
COHORT THAT FRANCIS MENTIONED IN
HIS INTRODUCTION AND ALL THE 
SLIDES I'LL SHOW FOR THE REST OF
THE TALK ARE ALSO FROM THE SAME 
COHORT. 
A GOOD NUMBER OF THEM ARE FROM 
BEGGUINEA PIG NUMBER ONE, WHICH
IS 
ME.
AND IT'S HAD ADVANTAGES.
TAKE TWO EXAMPLES HERE.
THE NIH HAS AN EN/KOCODE
PROJECT, 
WHICH IS AN ENCYCLOPEDIA DNA 
ELEMENT, WHICH IS AIMED AT 
OBSERVING THE
EP/SKWREPBIGENETICS IN 
EVERY WAY YOU CAN, LOOKING AT 
FACTORS, PROTEINS THAT BIND TO 
DIFFERENT BASE PAIRS THROUGHOUT 
THE GENOME, LOOKING AT RNA 
LEVELS AND SO FORTH.
BUT THE PROJECT WAS MOSTLY PRED
PREDICATED ON WHATEVER CELL 
LINES WERE CONVENIENT AT THE 
TIME AND SORT OF REPRESENTED 
VARIOUS TISSUE TYPES.
BUT THIS IS THE ONLY ONE THAT I 
KNOW OF WHERE WE HAD ISOJEGENIC 
SETS SUCH AS FIBROBLASTS WHERE 
THESE ALL HAD THE SAME GENOME 
BUT DIFFERENT EPIGENOMES.
SO I THINK THAT MADE A 
PARTICULARLY NICE SET AND THAT'S
PUBLICLY AVAILABLE, THE DATA 
FROM ALL THE LABS THAT WERE 
INVOLVED IN THE NIH CODE PROJECT
PROJECT.
ANOTHER PROJECT WAS A 
COLLABORATION OF THIS AND THE 
FDA.
THIS MAY BE THE FIRST TIME AND 
THEY ESTABLISHED GENOME IN /AA 
BOTTLE TO ESTABLISH STANDARDS 
FOR THE WORLD FOR PROVIDING 
GENOMES AND CELLS AND SO FORTH.
AND THEY LOOKED AROUND /THE
WORLD
FOR /AA COHORT THAT HAD BEEN 
PROPERLY CONSENTED FOR THIS SORT
OF THING AND OURS WAS THE ONLY 
ONE THAT HAD BEEN CONSENTED FOR 
POSSIBLE DISCLOSURE OF THEIR 
IDENTITY AND ALSO FOR COMMERCIAL
USE, BECAUSE THESE STANDARDS 
HAVE TO BE USED COMMERCIALLY.
SO ANYWAY, THIS PROJECT IS NOW 
INTERNATIONAL.
BOSTON, TORONTO, LONDON, VIENNA 
AND ALSO IN NEW YORK AND IN CASE
OF OFF TAUSTRIA, IT IS
SYNONOMOUS 
WITH THE ENTIRE INTERNATIONAL 
GENOME PROJECT.
IT'S CALLED GENOME OFF TAUSTRIA
AND 
IN COLLABORATION WITH MANY OTHER
OMICS.
AND ANY OF YOU ARE INVITED TO 
JOIN IT.
THIS IS AN -- ANOTHER EXAMPLE OF
USE OF THESE P /TKPW-FPG /P-FPLP
CELL LINE
S.
THIS IS FOR GETTING AT VARIANCE 
OF UNKNOWN SIGNIFICANT.
IT I WILLUSTRATES RUN IT ONE
/WAEWAY 
AND YOU HAVE GENE THERAPY AND 
YOU OTRUN ANOTHER ANOTHER WAY
AND
YOU GET A CAUSE AND EFFECT OF 
INDIVIDUAL POINT MUTATIONS DELL 
DID THE CLINICAL GENETICS ON 
THIS CARD /KWRO /PHAOIOMYOPATHY,
WHICH HE 
THOUGHT MIGHT BE A MUTATION IN 
THE TASS GENE ON THE X CHROME
CHROMOSOME OF THIS LITTLE BOY.
IT MIGHT BE THIS QUGUANINE.
NOW THE HYPOTHESES ARE NOT THIS 
NARROW.
THERE IS HUNDREDS OF POTENTIAL 
CANDIDATE ALLELES.
BUT THIS IS A NICE CASE.
SO THE X CHROMOSOME.
THERE IS ONLY ONE COPY OF THIS 
GENE.
AND ONLY ONE BASE PAIR.
SO THIS IS ONE BASE PAIR IN THE 
WHOLE 6 BILLION WE THOUGHT MIGHT
BE CAUSATIVE.
BUT UP TO /TTHAT POINT WE DIDN'T
EVEN HAVE CORRELATION DATA 
BECAUSE THIS WAS THE END OF ONE 
STUDY.
WELL, WHAT CAN YOU DO?
THE ANSWER IS QUITE A BIT ON 
THIS PARTICULAR CASE.
WE TURNED YOU IT INTO CAUSE AND 
EFFECT BY TAKING THESE BEGGUINEA
PIG NUMBER ONE CELL LINE, MAKING
IT -- PUTTING IN THE CRISPER, 
THE CAT HUMANIZED CAST /50EU69D 
ALONG WITH OUR PAIR.
IF YOU DON'T INCLUDE, YOU GET 
THIS MESS OF THAT SOME PEOPLE 
CALL EDITING BUT I CALL GENOME 
VALNDALISM.
AND BUT IF YOU DO INCLUDE THE RE
REPAIR, THEN YOU GET WITH FAIRLY
HIGH YIELD SORT OF IN THE 30 TO 
80% RANGE, DEPENDING ON THE 
TISSUE TYPE.
BUT YOU'LL GET A DELETION OF THE
GENE.
AN EXAMPLE OF THIS GENOME VALEN
VANDALISM IS AN /EUINSERTION OF
A 
RAND /O*OM BASE PAIRS THAT HAVE
NO 
MATCH, OBVIOUS.
ANYWAY, SO YOU CAN MAKE THIS ONE
ONE-GENE DELETION.
AND THEN YOU CAN MAKE
ORGANIOIDS.
SO YOU'VE DONE GENETIC CHANGE 
AND THEN YOU DO
EP/SKWREPIGENETIC RE
REPROGRAMMING AND THAT'S WHAT WE
HAVE HERE IS /THE ONE WITH 
PARKER.
WE'VE MADE CARDIAC MUSCLE HERE 
AND YOU CAN SEE THIS BEAUTIFUL 
REPEATING MOTIF OF THE 
ORGANIZATION OF THIS CARDIAC 
MUSCLE, WHICH /WILL CONTRACT 
NORMALLY WITH SORT OF A 6
/0-BEAT 
PER MINUTE RATE.
AND THEN ONE BASE PAIR IN THE 
WHOLE GENOME, JUST CHANGING THAT
ONE BASE PAIR CAUSES ABNORMAL 
BIOKCHEMISTRY, MORPHOLOGY AND 
FIPHYSIO/HROPBLLOGICAL BEATING.
AS DOES INSERTING EIGHT BASE 
PAIRS.
THEY ARE BOTH OUT /OF FRAME FOR 
THE TASS PROTEIN.
NOW YOU MIGHT SAY WELL, YOU'VE 
TALKED ABOUT OFF-TARGETS AND ON 
TARGET PROBLEMS WITH CRISPER.
HOW DO WE KNOW THAT THIS IS 
REALLY WHAT YOU SAY IT IS?
AND ANSWER IS THAT MY GRADUATE 
STUDENTS THINK NOTHING OF WHEN 
THEY CHANGE ONE BASE PAIR OF 
SEQUENCING ALL 6 BILLION BASE 
PAIRS TO MAKE /SHAOSURE THEY DID
THE 
RIGHT THING BECAUSE IT IS IN
INEXPENSIVE NOW.
AND THAT MIGHT BE A LESSON THAT 
SOMEDAY WE'LL THINK NOTHING OF 
SIN /TYNTHESIZING ALL 6 MILLION
BASE 
PAIRS JUST TO CHANGE IT IF IT'S 
CHEAP ENOUGH.
STRANGER THINGS HAVE HAPPENED.
IN ANY CASE, THIS IS KIND OF 
PROOF IN /AA RO1 STUDY THAT YOU 
HAVE CAUSE AND EFFECT OF THAT 
SINGLE QUGUANINE FOR THAT CARDIO
MI/YOPATHY.
SO BACK TO BRAINS.
FIRE MOMENT HERE.
WE ARE NOT JUST INTERESTED -- 
FOR /AA MOMENT HERE.
WE ARE NOT JUST INTERESTED IN 
MAKING BRAINS.
WE ALSO WANT TO BE ABLE TO MAKE 
SURE THAT WHEN WE MAKE THESE 
ORGANIOIDS, THEY ARE FIZPHYSIO
PHYSIO/HROPBLOGICALLY REASONABLE
AND 
IF THEY ARE NOT, WE WANT /TO DE 
BUG THEM WITH ALL THE TOOLS WE 
CAN AND WE CALL THIS A THROUGH E
ACTIVITY, WHICH IS TYPICALLY 
DONE WITH CASTLCIUM IMAGING.
BUT WE CAN -- WE ARE DEVELOPING 
METHODS WHERE WE CAN DO IT IN C
SITU AND TIME DOES NOT PERMIT.
BUT QUESTIONS CAN ASK.
THEN WE MONITOR BEHAVIOR YOU IN 
THE CASE OFTEN VIDEO IN/PPUT FOR
VISUAL TASS AND VIDEO OF WHAT 
THE RESPONSE, MOTOR RESPONSES 
ARE.
THEN THE CONNECT I'LL HAVE AND 
HOW THE SYNAPSES ARE CONNECTED.
THE DEVELOPMENTAL LINEAGE.
HOW EACH CELL IN THE BODY GOT IT
WHERE IT WAS FROM THE FERTILIZED
EGG AND FINALLY EXPRESSION 
ANALYSIS, SO RNA PROTEIN.
THIS IS FUNDED BY IRPA AS PART 
OF THE BRAIN INITIATIVE AND 
THESE ARE SOME OF MY COLLEAGUES 
AT HARVARD AND COLUMBIA.
AND MITT.
AND THE MAIN THING IS GETTING 
THIS CONNECT OOME AND
CORRELATING 
IT WITH THE ACTIVITY MAP WITH 
CASTLCIUM.
THIS IS -- THIS AND THE 
ESSENTIALLY ALL OF THE THINGS ON
OUR A THROUGH E LIST ARE HELPED 
BY THE FACT THAT NEXT GENOME 
SEQUENCING IS ACTUALLY AN 
IMAGINING METHOD.
WHEN WE FIRST DEVELOPED IT IN 19
1999, WE LITERALLY USED A MICRO
MICROSCOPE.
BUT CONVENTIONAL SEQUENCE UGRAND
UGRANDOMIZE THE CELL CONTENTS 
AND DISPLAY THEM ON A FLAT 
SURFACE.
THE ORIGINAL CONCEPT, EVEN 
BEFORE NEXT GEN SEQUENCING, NOW 
CALLED CONVENTIONAL SEQUENCING 
AND WHAT WE HAVE NOW IMPLEMENTED
IMPLEMENTED, CALLED INSITU 
SEQUENCE, IS TO LEAVE THE CELLS 
ALONE ON A SURFACE.
THEY CAN BE FAIRLY THICK.
TENS OF MICRONS THICK, AND 
SEQUENCE THEM WHERE THE RNAS AND
DNAS AND PROTEINS LIE IN THE 
FIRST PLACE.
BUT IN BOTH CASES, IT'S JUST A 
SERIES OF CYCLES WHERE YOU USE 
FOUR COLONS -- CODONS PER CYCLE 
AND YOU BUILD UP AND GET AN 
INFORMATION CONTENT OF 4 TO THE 
N, WHERE N IS /THE NUMBER OF 
CYCLES AND IT'S EASY TO DO, 
HUNDREDS OF CYCLES WITH NEXT GEN
SEQUENCING, WHETHER IT'S IN SITU
OR CONVENTIONAL.
WE TEND TO AIM FOR 30 CYCLES OR 
LESS, BECAUSE YOU CAN GET ALL 
THE INFORMATION YOU NEED FROM A 
SHORT TAG.
NOW I AM GOING IT SHOW YOU MULTI
MULTICELL /PHRAURLT SEQUENCING 
AND LATER SUBCELLULAR, BECAUSE R
RNAS AND PROTEINS ARE NOT 
UNIFORMLY DISTRIBUTED AND YOU 
CAN'T TAKE THEM OUT AND ANALYZE 
SINGLE CELLS.
YOU HAVE TO SEE THEM IN CONTEXT.
AND WHERE THERE IS FOUR COLORS, 
EACH NEURONUR -- NUR /O*ON HAS A
SPECIFIC
COLOR BECAUSE WE'VE BAR CODED 
THE ENTIRE NUR /O*ONS.
THERE WILL BE THOUSAND /S OF
DOTS 
BUT FOR THIS PURPOSE, WE ARE BAR
CODING THESE NURONS SO WE KNOW 
HOW /TO CONNECT OVER SOMETIMES 
CENTIMETER DISTANCES AND THIS IS
WORK FROM TONY ZAIDER'S GROUP.
WATCH ON THE SIDE WE ARE MONITOR
MONITORING ONE AND TWO AND 
BUILDING APE BAR CODE.
AND THE CELLS, ALL THE PROCESSES
WILL STAY IN PLACE BUT THE 
COLORS WILL CHANGE.
AND YOU CAN SEE ALL THE AX
/O*ONS 
AND DENDRITES AND YOU CAN SEE 
THE BAR CODES BUILDING UP ON THE
SIDE.
NOW THIS IS SUBCELLULAR IN SITU 
SEQUENCING.
THIS IS EACH DOT THERE IS A SING
SINGLE RNA MOLECULE -- MOLECULE 
AND THE DOTS STAYED IN PLACE BUT
THE COLORS KEPT CHANGING.
AND THEN WE CAN RECORD ALL THOSE
COLORS AND PLAY THEM BACK OUT OF
THE PLANE AS /AA BAR CODE.
SO EVERY POSITION IN THERE HAD A
BAR CODE, WHICH WAS BUILT UP THE
SAME WAY NEXT GEN SEQUENCING, 
EXCEPT WE DIDN'T BOTHER TO THROW
AWAY THE MORPHOLOGY AND THE 3-
3-DIMENSIONAL COORDINATES OF 
EACH OF THE RNAS.
BUT WE CAN ALSO USE DNA AND 
PROTEIN, AS YOU CWILL SEE IN THE
NEXT COUPLE OF SLIDES.
AND WE CAN DO SOUPER RESOLUTION 
AS WELL.
ONE IS CALLED STORM, WHICH IS 
FIPHYSICS AND OPTICS STRATEGY 
WHERE YOU QUERY OVER AND OVER 
THE SAME FAILULUOROFLORA AND YOU
GET
A BETTER AND BETTER CENTROID FOR
IT.
SO HERE IS CONVENTIONAL MICROPSY
AND THE SAME MAGIFICATION AND 
GETTING THE 5 TO 20 NAN /OMETER 
RESOLUTION.
AND SO IT'S ON THE ORDER OF NUKE
NUCLEOSOMES AND THIS IS SWEEPING
THROUGH THE FIELD.
IT'S WORK FROM PROFESSOR AT 
HARVARD MEDICAL SCHOOL.
SO THAT'S RNA AND DNA.
NOW PROTEINS SEEMS A LITTLE HARD
HARDER.
AND IT IS.
BECAUSE WE'RE LIMITED BY ANTI-
ANTIBODY RESOURCES.
BUT IF YOU HAVE THESE PURPLE 
PROTEINS AND GRAY ANTIBODIES, 
THESE Y-SHAPED MOLECULES, TAGGED
WITH A NUCLEOTIDE, COUPLED TO A 
POLL MER.
SO STILL STRAITS TWO THINGS.
ONE IS HOW /TO TAG PROTEINS AND 
TURN IT INTO A NUCLEIC ACID 
PROBLEM.
AND THE OTHER IS HOW /TO GET A
MI
MICROP /SP*EU WHERE YOU LITERAL
LITERALLY TAKE THE CELL AND EX
EXPAND IT BY -- THIS IS POLYMER 
ACRYL AATE, THE ACTIVE
INGREDIENT 
OF DISPOSABLE DIAPERS, WHICH /HR
WILL ABSORB A LOT OF WATER.
IF YOU DO ONE ROUND OF EXPANSION
AND 100 SQUARED.
AND SO FOR EXAMPLE, HERE IS A 
PRE/S- AND
POST-SIGNIFICANYNAPTIC PROTEINS
 
THAT ARE NORMALLY NOT RESOLVED 
THAT ARE BEAUTIFULLY RESOLVED 
AND HELP US TO DETERMINE WHERE 
THE SYNAPSES ARE AND WHICH 
DIRECTION THEY GO.
AND THIS IS WORK THAT WE'VE DONE
FOR /AA FEW YEARS NOW WITH ED
BOYD
BOYDEN'S GROUP.
NOW, INTEGRATED WITH EXPANSION 
THAT'S CALLED EXPANSION SEQUENCE
SEQUENCING AND EXPANSION PHISH, 
FOR WAYS INTEGRATED WITH OTHER 
NUCLEIC ACIDS.
THIS IS A BUSY SLIDE, BUT THE 
TAKE-HOME HERE IS WE HAVE NINE 
DIFFERENT WAYS, WHICH IS LIKE 
NINE HE HEDITORS, DIFFERENT WAYS
OF 
DETECTING METABOLITES WHERE WE 
CAN ESSENTIALLYTARIAN STILL 
AHEADY METABOLITE LEVEL INTO A 
PROTEIN LEVEL BY STABILIZING 
THAT PROTEIN.
AND YOU HAVE ALREADY SEEN THAT 
WE CAN DETECT PROTEINS IN SITU.
SO WE ARE PURSUING THIS AS /AEA
WAY
OF DOING MET BLOMIC S S IN SITU.
IN SUMMARY WITH THAT PART WE 
HAVE OMNIOMICS, WHICH IS PRETTY 
COOL BY ITSELF.
BUT YOU ADD THE WORD IN SITU TO 
IT AND THEN YOU CAN DO PROTEOME 
S IN SITU, WHERE YOU HAVE ALL 
THE ADVANTAGES OF MY CROSSPI AND
MORPHOLOGY.
SO THE LAST THING -- AND I THINK
WE ALL SHOULD DO AND MARSHAL DID
IS TO ENGAGE WITH SOCIETY AS A 
WHOLE.
THINK ABOUT WHERE THIS IS ALL 
GOING AND CERTAINLY MANY OF THE 
TECHNOLOGIES I HAVE BROUGHT UP 
RAISE ISSUES.
AND SINCE THE TECHNOLOGISTS SEE 
THEM FIRST, WE HAVE A SPECIAL 
RESPONSIBILITY OF POINTING OUT 
THE DOWN /SKPAOEUTDZ SOLUTIONS 
TO THE DOWN SIDES AND THEN THE 
PROBLEMS THAT THE SOLUTIONS 
CREATE AND THEN THE FURTHER 
TWEAKS, WHICH MAKES PEOPLE 
NERVOUS, BUT WE NEED TO DO IT.
SO FIRST THING I'LL JUST JUMP 
RIGHT TO WHERE IS EVERYBODY AS 
GENETICALLY MODIFIED HUMANS.
THERE ARE ALREADY TENS OF 
THOUSAND /S OF GENETICALLY 
MODIFIED HUMANS BECAUSE 2300 
CLINICAL /TRAOEUTRIALS OVER
/-THE YEARS 
FOR GENE THERAPY.
YOU MAY NOT SAY THAT'S NOT A 
GENETICALLY MODIFIED HUMAN BUT 
IS /* IT IS.
ONE /OF THEM IS APPROVED AND THE
DELIVERY VEHICLE FOR THAT IS 
ASSOCIATED VIRUS, WHICH EXPLAINS
WHY SO MANY PEOPLE ARE EXCITED 
ABOUT A AD BECAUSE IT'S APPROVED
AND IT'S FAIRLY BENIGN IN EVERY 
WAY.
BUT IT'S REALLY TERRIBLY SMALL.
IT'S TERRIBLE IN THAT REGARD.
IT IS HOPEFULLY THE PRICE WILL 
DROP.
AS WITH ORPHAN DRUGS, YOU HAVE 
ALMOST THE SAME COST OF 
DEVELOPMENT AS /AA REGULAR DRUG 
BUT A SMALLER MARKET.
AND FOR.
THESE GENETIC COUNSELING IS AN 
ALTERNATIVE.
OKAY SO YOU ARE SAYING THAT'S 
NOT REALLY GENETICALLY MODIFIED 
HUMANS.
IT'S ABOUT THERAPY.
THERE ARE ALREADY PEOPLE WHO 
HAVE RECEIVED MITOCHONDRIAL DNA 
THERAPY.
SOMEHOW THAT ISN'T ALWAYS 
CLASSIFIED AS THERAPY.
I THINK IT SHOULD BE.
THERE IS A POSSIBILITY OF USING 
EXPERIMENTIC STEM CELLS, WHICH 
IS NOT ALWAYS DISCUSSED.
TYPICALLY THIS IS ABOUT EMBRYOS,
WHICH IS PROBLEMATIC BECAUSE OF 
MONTH SAKEISM, WHERE DIFFERENT 
CELLS HAVE DIFFERENT TYPES AND 
SO YOU CAN'T REALLY DETERMINE 
WHETHER YOU ARE CRISPER OR OTHER
GENE THERAPY WORKED.
BUT WITH STEM CELLS YOU CAN GET 
CLOSE TO 100% CORRECT EDITING BY
THE METHOD I DESCRIBED EARLIER 
FOR THE CARDIAC STUDY BY ANALYZE
ANALYZING STEM CELL CLONES.
AND I THINK IF -- THIS WILL DEAL
WITH /EUINFERTILITY BUT ALSO IF 
IT'S APPLIED TO /RECESSIVE
GENES,
SUCH AS T SACKS, WHICH ALREADY 
ARE BEING TREATED BY EITHER 
ABORTION OR IBFPGD, WHICH SOME 
WOULD CLARISSIFY WITH AS EVEN
MORE
EMBRYO AT RISK, YOU MIGHT HAVE 
THAT IDEA OF P /TKPW-FPGD AND
80% AT RISK
WITH IT.
SO THE IRONY IS THAT GERM-LINE 
THERAPY MIGHT REDUCE EMBRYO HARM
RATHER THAN INCREASE IT.
SO I THINK WE NEED TO THINK, BE 
NOT TOO DIFFERENCE? IN WHEN WE 
HAVE TO THINK CAREFULLY ABOUT 
THIS.
-- DISMISSIVE.
AMP
AMPING IT UP FURTHER IN TERMS OF
ALARM LEVEL.
THERE IS THIS TISSISSUE OF CAN
WE 
ALTER COG /TPHENITIVE RATES?
DISMISSING IS NOT A HEALTHY 
THING FOR EITHER SIDE.
BECAUSE IT'S HARD TO SEE IT IN 
-- VARYING WITH ALLELES IN 
NATURAL POPULATIONS.
BUT IN SYNTHETIC SITUATIONS, 
JUST LIKE YOU WILL SEE A /* BIG 
IMPACTS -- SORRY, IN RARE CASES 
YOU WILL FIND BIG IMPACTS AND 
ALSO IN SYNTHETIC CASES IN A 
MOMENT.
FOR EXAMPLE, EFFECTS ON AL
ALZHEIMER'S AND SOME THAT AFFECT
DISABILITIES.
BUT THEN THERE IS GENE THERAPY 
TRIALS THAT ARE BASED ON SOME
SOMEWHAT ABNORMAL, NOT FOUND IN 
HUMAN POPULATIONS, BUT BASED ON 
GENES THAT ARE IN THE GENOME.
AND SO FOR EXAMPLE, THERE ARE 
SOME GENE THERAP TRIALS AND 
EITHER PRECLINICAL ANIMAL OR 
HUMAN TRIALS.
AND THERE ARE EVEN SOME THAT 
SIGNIFICANTLY ENHANCE 
PERFORMANCE AND COG /TPHENIT 
/STKPWHRAOEUFRPBLGSZ SHOWING THE
BLACK ONES, WHERE YOU HAVE A 
SIGNIFICANT INCREASE.
VERY SPECIFIC COG /TPHENITIVE
TASKS.
MAYBE SOMETIMES MULTIPLE ONES.
AND THE REASON THAT WE MIGHT NOT
BE ABLE TO DISMISS IT AS MULTI
MULTIGEMULT
MULTIJGENICS AND COMPLEX,
MEANING
SO MANY GENES IN ENVIRONMENTAL 
COMPONENTS IS THAT EVEN THOUGH 
THERE ARE MAYBE 10,000 SINGLE 
NUCLEOTIDE MORPHISM THAT'S 
AFFECT THINGS LIKE HEIGHT, THE 
EXAMPLE OF /AA COMPLEX STRAIGHT 
WITH GENETIC ENVIRONMENTAL 
COMPONENTS.
IT'S NOT ALL ABOUT THE NATURAL 
VARIATION IN THE BELL CURVE.
IT'S ABOUT THE ENDS OF THE BELL 
CURVES AND AT THE END ARE THINGS
THAT AFFECT GROWTH HAORM ONONE
AND 
RECEPTORS.
AND WHEN YOU FIND THOSE, THEY'RE
VERY OFTEN NOT ONLY
IN/TPORPFORMATIVE 
ABOUT THE PATHWAYS, BUT ALSO 
THEY PROVIDE THERAPIES.
AND HERE IS AN /KPW-RL GROWTH 
HA
HORM OONE THERAPYS WORK ON
ALLELES
AND HERE IS AN /KAEUINDICATION
WHERE 
GROWTH HORMONE IS USED AS /AA
DRUG
DRUG.
EVEN MORE ALARMING IS PEOPLE -- 
SO WE'VE GONE FROM GENETICALLY 
MODIFIED HUMANS TO GERM-LINE TO 
COMPLEX TRAITS.
TO NOW DO IT YOURSELF GENE 
THERAPY AND THIS IS A REAL THING
THING.
IT'S BEEN /ROREPORTED IN TWO 
ARTICLES IN "TECHNOLOGY REVIEW,"
AMONG OTHERS.
I CAN GIVE THEIR NAMES BECAUSE 
THEY ARE PUBLIC.
AND THEY ARE DO IT YOURSELF 
CRISPER KITS FOR $150, ASSUMING 
NO PRIOR EQUIPMENT WHATSOEVER.
AND THESE TWO DID GENE THERAPY 
BY OUTSOURCEING IT.
ONE /OF THEM DOESN'T HAVE ANY 
BIOLOGICAL TRAINING WHATSOEVER.
THESE WERE TESTED IN ANIMALS BUT
NOT EXACTLY THE SAME GENE 
THERAPIES.
SO AND IN /THIS CASE I DON'T 
THINK IS A GOOD THING.
THE OTHER ONE THAT I MENTIONED 
EARLIER IS FINE.
BUT THESE WERE TESTED IN ALL 
THESE DIFFERENT ANIMALS.
AND THEY ARE TRYING TO REVERSE 
AGING AND IT'S NOT COMPLETELY 
OUT TO LUNCH TO DO THAT.
THERE IS A GREAT DEAL OF 
INFORMATION KNOWN ABOUT AGING 
AND MODEL ORGANISMS.
AND IN FACT, OUR GROUP HAS TAKEN
45 OF THESE INTO GENE THERAPIES 
FOR VET NEAR PRECLINICAL 
/STKWRAOEURBLGSZ COULD THEN LEAD
TO HUMAN.
AND IN FACT THE FDA PROCESS FOR 
VET NEAR IS MUCH FASTER.
AND I THINK THIS IS A MORE 
CAUTIOUS APPROACH.
BUT WE'LL SEE.
ANOTHER APPLICATION, WHICH IS /*
-- WHICH HAS CAUSED SOME CONCERN
15 YEARS AGO THERE WAS CONCERN 
THAT TRANSPLANTING PIG ORGANIS 
INTO HUMANS WOULD IN ADDITION TO
IMMUNE PROBLEMS, WOULD HAVE IN
IN/TKOPBENDOGENOUS
RE/TRTROVIRUSES THAT 
WILL INFECT HUMAN CELLS AND IT 
IS KNOWN THAT THEY DO INFECT 
HUMAN CELLS.
AND SO ESPECIALLY IMMUNE COM
COMPROMISED HOST WOULD NOT BE 
GREAT.
SO WHEN WE BROUGHT CRISP TORE 
BEAR ON THIS PROBLEM 15 YEARS 
LATER AND IT WAS ABOUT A $15 
BILLION INVESTMENT AND IT 
DROPPED BECAUSE OF PER VS DISH 
DIDN'T COME UP WITH THE
AKRCRONYM 
-- WE KNOCKED OUT ALL 62 AT ONCE
AND IT WAS NOT HEROIC.
14 DAYS IN AN INK /SKPWAEURT 
LITTLE BIT OF PCR IS ALL IT WAS.
AND WE KNOCKED OUT MOST OF THESE
45 GENES THAT WERE INVOLVED IN 
THE CLOTTING IN/KPABTS WITH 
HUMANS.
SO NOW THESE WERE FETUSES.
THIS IS NOT PUBLISHED.
WE PUBLISHED THE INSIDE
DODGENOUS
RE/TTROVIRUS, WHICH AS A AS A 
RECORD FOR CRISPER 62 AT A TIME 
WHERE TWO OR THREE GENES AT ONCE
WAS CHALLENGING.
WE HAVE POSITIVE ULTRASOUNDS, 
BUT I WON'T BELIEVE IT UNTIL THE
PIGLET ARE HEALTHY AND ARE USED 
TO TRANSPLANT TO -- INTO NON-
NON-HUMAN PRIMATES.
HOPEFULLY LATER THIS /KWRAYEAR.
AND A /HRWILL THE OF THIS WAS
LED 
BY YANG, WHO WAS A POST DOC AND 
IS NOW LEADING THIS COMPANY 
CALLED EGENESIS.
ANOTHER TROUBLING -- POSITIVELY 
AND NEGATIVELY -- EACH OF THESE 
HAS VERY POSITIVE COMPONENTS AS 
WELL -- IS THIS IDEA OF GENE 
DRIVES.
THIS COULD BE A REAL BOON TO 
PUBLIC HEALTH, ESPECIALLY IN 
COUNTRIES THAT CANNOT AFFORD 
OTHER MEDICINES AND EVEN VAX
VACCINES ARE HARD TO DISTRIBUTE 
INTO REMOTE AREAS OR WAR ZONES.
HERE YOU CAN AIM IT TO 
/SKPRERBGTS THE VECTORS SPREAD 
-- SPREAD THE GOOD PACKAGE 
THEMSELVES AND THE PACKAGE IN 
THIS CASE MIGHT BE ANTI-MA
ANTI-MA/HRAEANTI-MALARIAL
MULTIPLE ANTI-MA/HRAELARIAL
PEPTIDES 
OR ANTIBODIES THAT SPREAD 
THROUGH THE MARRIAGE CERTIFICATE
POPULATION -- MOSQUITO 
POPULATION, NOT AT THE NORMAL 
RATE OF 50%, WHICH DOESN'T 
REALLY CHANGE.
BUT AT 100%.
AND HERE IS AN EXAMPLE OF /AA 
STANDARD DRIVE.
AND ONE OF THE THINGS WE DID IN 
THIS CASE IS WE GOT AHEAD OF THE
GAME AND TALKED ABOUT THE SAFETY
ISSUES AND THE REVERSEIBILITY 
AND KEEPING THINGS LOCAL BEFORE 
WE STARTED THE /SPEURPLTS RATHER
THAN ANNOUNCEING IT LIKE GAIN OF
FUNCTION FLU AFTER WE ARE ALL 
DONE.
AND THEN WE DID THE EXPERIMENTS 
IN /AA VARIETY OF ORGANIZSMS
RANGE
RANGING FROM YEAST TO /THE
L/-THE MA
MA/HRAELARIA IN MOSQUITOES.
AND IDEA IS THAT CRISPER CUTS, 
YOU HAVE -- IN AN ESSENTIAL AND 
GREEN TRANSFERS OVER TO A COPY 
OF THE GENE DRIVE, WHICH 
INCLUDES THE WHOLE CRISPER 
MACHINERY, AS WELL AS YOUR PAY 
LOAD, WHICH /WILL FIGHT THE MA
MA/HRAELARIA AND MAKE /-HE
MOSQUITO RE
RESIST AANT.
AND IT IN /AA STANDARD DRIVE IT 
GETS CUT AND REPAIRED AND NOW 
YOU HAVE TWO COPIES SO ALL THE 
OFFSPRING, 100%.
AND THIS IS BOTH THE CALCULATED 
AND OBSERVED RATE OF SPREAD, AS 
IT GOES TO FIXATION.
AND WE'VE COME UP WITH A DISAISY
DRIVE.
I'M NOT GOING TO GO THROUGH THE 
DETAILS, UNLESS YOU WANT TO IN 
THE QUESTIONS, WHERE CRISPER ONE
CUTS CRISPER TWO.
AND CRISPER TWO CUTS A AND THEN 
ALL DECAY BECAUSE THERE IS A 
SLIGHT DISADVANTAGE IN /THIS 
MODEL.
BUT A GOES CLOSE TO FIXATION AND
COULD EVEN GO TO FIXATION, 
DEPENDING ON HOW MANY STAGES OF 
CRISPER AND HOW YOU RIG THIS.
BUT IT CAN CONTROL THINGS IN 
TIME AND IN SPACE.
THIS IS AGAIN IN /THIS LOVELY 
JOURNAL CALLED "BIOARCHIVE."
SO IN WRAPPING UP, I WANT TO SAY
THREE THANK YOUS.
ONE IS TO P /TKPW-FPG ED.ORG FOR
THE 
/SKPHOEPBT EDUCATION OF THIS.
THEY DO CONGRESSIONAL BRIEFINGS 
WITHOUT LOBBYING QUARTERLY.
THEY REACH OUT TO MINORITY HIGH 
SCHOOLS AND WORK WITH SCREEN 
WRITERS AND THEN THEY HAVE THESE
ONLINE GAME TOOLS LIKE MAP ED.
AND THIS IS INITIATED BY TEAM 
WOO IN 2006.
AND JANETIN IS AN ETHEXIST HAS 
BEEN WORKING WITH US EVER SINCE 
OUR FIRSTER GRANT AND HAD AN LC 
COMPONENT IN IT AND WE'VE 
PUBLISHED MANY PAPERS TOGETHER, 
ABOUT 20 OR SO.
AND FINALLY I WANT TO THANK THE 
NIH FOR FUNDING OUR CENTERS OF 
EXCELLENCE FOR GENOMIC SCIENCE.
IN THIS CASE, OUR THIRD ON THREE
DIFFERENT TOPICS.
FIRST ONE WAS NEXT GEN SEQUENCE
SEQUENCING IN 2004.
THEN ONE ON ZINC FINGERS, WHICH 
WE DIDN'T DELIVER BUT WE DID 
DELIVER CRISPER.
SO I FIGURED THEY WERE OKAY WITH
THAT.
AND ALSO ON THE FIRST ONE WE 
DIDN'T DELIVER THE 1.7 BASE PAIR
BACTERIAL GENOME THAT WE PROMISE
PROMISED BUT DELIVERED 5 MILLION
GENOMES IN NEXT GEN SEQUENCE.
SO WE'RE GETTING AWAY WITH 
MURDER ON THESE GRANTS.
BUT THE NEXT ONE IS ON ORGANIS.
AND I MENTIONED THE I ORPA 
PROJECT AND DIDN'T MENTION TWO 
TRANSFORMATIVE AWARDS FOR 
MOLECULAR RE/* RECORDING AND 
USING THIS IN SITU METHOD ON 
HUMAN SAMPLES.
SO QUESTIONS?
THANK YOU.
/PHRA
[APPLAUSE]
>>  SO PLEASE GO TO THE 
MICROPHONES TO POSE QUESTIONS SO
THAT EVERYBODY WATCHING ON VIDEO
CAN HEAR.
WHILE THEY ARE THINKING ABOUT IT
IT, GEORGE, LET ME ASK.
I WAS VERY STRUCK WITH THIS 
EFFORT TO FIND EXPRESSABLE 
VERSIONS OF ALL THE 
TRANSCRIPTION FACTORS AND THAT 
YOU ARE SEEING SOME PRETTY 
SURPRISING THINGS.
AND OBVIOUSLY ONE WANTS TO GO 
FROM SINGLE FACTORS TO MIXING 
THEM UP /SAND IF YOU WERE GOING
TO
DO TWO AT A TIME, THAT'S 2 
MILLION /123ER789S.
IS THAT FEASIBLE?
>>  RIGHT.
SO WHAT WE'RE DOING WITH A SING
SINGLE FACTORS THROUGH THE 
LIBRARY IS WE WILL TAKE A TARGET
TARGET, LET'S SAY, AND DO RNA 
SEQUENCIG ON SINGLE CELLS SO WE
KNOW WHAT THE LEVELS OF THE 
TRANSCRIPTION FACTORS ARE.
WE TAKE THE TOP ONES, AND WE PUT
THOSE IN /AS /AEA MIXTURE SO
WE'RE 
NOT DOING FULL.
AND THEN WE'LL PUT IN THE WHOLE 
LIBRARY AS THE UNPROGRAMMED PART
IN CASE WE MISSED SOMETHING OR 
THERE IS SOME THING SYNTHETIC 
THAT CAN HELP US OUT.
THAT'S OUR CURRENT PLAYS.
BUT WE WANTED TO KNOW WHAT THEY 
ALL DID INDIVIDUALLY SO WE CAN 
PUT THAT INTO THE MODEL.
PAIRS, WE CAN PROBABLY DO THE 
WHOLE PAIR-WISE.
TRIPLES I THINK IS GETTING OUT 
OF RANGE AND THAT'S WHERE YOU 
USE THIS OTHER METHOD WHERE YOU 
PUT IN AS MANY AS YOU CAN GUESS 
AT AND THEN PUT IN THE WHOLE 
LIBRARY.
>>  MAKES SENSE.
OVER HERE.
>>  FROM PAST EXPERIENCE IN PED 
PEDIA TRICK PATHOLOGY I CAN TELL
YOU THAT THE SOXIX CAN RE
REGENERATE ALL OF THE ORGANIS
AND
ESSENTIALLY ALL OF THE TISSUES 
BECAUSE I'VE SEEN A FEW OF THEM.
I CAN TELL YOU THAT IT'S ALWAYS 
A QUESTION WHY YOU WOULDN'T JUST
WANT /TO INJECT SOME STEM CELLS 
INTO PEOPLE.
GENERATE IT.
AND MY UNDERSTANDING IS THAT YOU
PUT STEM CELLS TO A CRITICAL 
MASS AND THEY WILL AUTOMATICALLY
CONVERT TO A TOMA AND YOU CAN 
PICK THE CELLS OUT THERE HAVE TO
DO YOUR ENGINEERING RATHER THAN 
TO GO THROUGH ALL OF THIS PLURI
PLURIPOTENT STEM CELL ET CETERA.
IT SEEMED THAT YOU HAVE 
PERFECTLY VIABLE NEUROTISSUE.
YOU HAVE ALL THE TISSUE -- SKIN 
TISSUE.
SO THERE IS THERE ANY ANSWER TO 
THAT OR IS THAT JUST /AEUNOT A 
VIABLE IDEA?
>>  YES.
THAT'S A GREAT QUESTION.
IN FACT, I STARTED MY WORK WITH 
GAIL MARTIN, WHO WORKED IN TERA
TERATOMAS IN THE 80S.
I DO /* DID MY POST DOC IN HER 
LAB IN 198 /1K35* SHE WAS ONE OF
THE FIRST PRACTITIONERS OF EM
EMBRYONIC STEM CELL MANIPULATION
MANIPULATIONS.
BACK BEFORE IT WAS POPULAR.
THE -- WHAT WE'RE TRYING TO DO 
IS WE'RE TRYING TO MAKE 
ORGANIZED ORGANIS, NOT JUST 
TISSUES.
WE WANT /TO KNOW THE RULES AND
WE
WANT /TO KNOW WHAT WE CAN DO AND
WE WANT -- THE CLOSER THE ARTS 
OF FIPHYSIO/HROPBLLOGICAL
ORGANIZS.
SO THE TERATOME /KWRAAS FAR FROM
MORPH
MORPHOLOGY.
THEY WILL HAVE LOCAL PIECES THAT
ARE QUITE INTERESTING BUT THEY 
DON'T HAVE THE RIGHT -- SO 
THAT'S OUR GOAL.
AND I SHOWED YOU AN EXAMPLE 
WHERE WE'RE VAS /KHCLAIZING AND 
PUTTING IN GLIAL CELLS INTO THE 
PREVIOUSLY FAIRLY SUCCESSFUL 
CEREBRAL ORGANIOIDS, WHICH WERE 
INSTRUMENTAL IN FIGURING OUT MI
MI/KCROSELCEPHALY.
BUT A /HRWILL THE OF THIS HAS TO
DO
WITH THE BASIC RESEARCH OF 
FIGURING OUT HOW ORGANIZS
ASSEMBLE
ASSEMBLE.
>>  OKAY.
THANK YOU.
>>  IT'S AN UNUSUALLY SHY 
AUDIENCE.
DON'T BE SHY, FOLKS.
THERE ARE A LOT /OF ISSUES WE
CAN
TALK ABOUT WHILE THEY'RE 
THINKING.
YOU SHOWED THE DATA ON WHAT
/UYOU 
ARE DOING IN TERMS OF PREPARING 
PIGS TO BE ABLE TO PRODUCE 
ORGANS THAT WOULD BE ACCEPTABLE 
TO HUMANS BOTH BECAUSE YOU HAVE 
KNOCKED OUT THE
IN/TKOPBENDOGENOUS VIRUS
VIRUSES AND TRIED /TO MAKE THEM 
LESS ENDOJGENIC.
OBVIOUSLY, THE ALTERNATIVE, 
WHICH OTHER PEOPLE ARE PURSUING,
IS /THE IDEA OF MAKING CHIMEE 
ERICK EMBRYOS, WHERE YOU END UP 
WITH A PURELY HUMAN ORGANI IN A 
PIG HOST BECAUSE THE PIG IS 
ENGINEERED SO THAT IT CANNOT 
MAKE ITS OWN HEART BECAUSE OF 
THE PAPER DESCRIBING THIS.
SO WHAT IS YOUR VIEW HERE OF THE
PROS AND CONS OF OTHOSE A
APPROACHES, BOTH OF WHICH HAVE 
ETHICAL ISSUES THAT ARE QUITE 
INTERESTING AND VEXING?
>>  SO I WOULD ADD A THIRD A
APPROACH, WHICH IS MAKING HUMAN 
ORGANIS ENTIRELY OUTSIDE OF AN
AN
MAL.
BUT LET'S TALK ABOUT THOSE TWO.
THE PROBLEM WITH THE CHYM /ERAS 
-- FIRST OF ALL /SKWR-RB, IT'S
ETHICALLY 
MORE PROBLEMATIC.
BUT IN ADDITION TO /TTHAT, WHEN 
YOU TAKE OUT ONE ORGANI, YOU RE
REMOVE IT FROM THE DEVELOPMENTAL
POTENTIAL, AND THERE IS 
ESSENTIALLY A GAP FILLED BY THE 
HUMAN STEM CELLS.
YOU REALLY TYPICALLY HAVE GOTTEN
RID OF A SMALL NUMBER OF CELL 
TYPES AND THERE ARE MANY OTHERS 
THAT ARE STILL IN THERE -- 
NERVES, CAP /HRILLARIES, VARIOUS
MUSCLE TYPES, SMOOTH MUSCLE, ET 
CETERA -- AND IF YOU DON'T RE
RE/PHRAEPLACE ALL OF THEM, THEN
YOU 
ARE GOING TO HAVE A HOST RE
REJECTION OF THAT PART.
AND THAT'S GOING TO RESULT IN NE
NECROSIS PROBABLY.
SO AND MOST OF THESE HAVE BEEN 
OVER A /* VERY SHORT DISTANCES 
LIKE RAT-TO-MOUSE KIND OF 
DISTANCES.
AND I AM NOT SAYING IT'S /EUPL
IMPOSSIBLE.
THESE ARE SOME OF THE LIMITATION
LIMITATIONS.
SO IF YOU ARE GOING TO HUMANIZE 
THEM SO THAT THEY CAN TAKE ON --
ENGINEER THEM TO TAKE ON HUMAN 
CELLS, YOU MIGHT AS WELL HUMAN
HUMANIZE THE WHOLE THING BECAUSE
THE THIRD PROBLEM IS THAT IF YOU
HAVE -- IF YOU HAVE ENGINEERED 
ONE ORGANI, SAY THE HEART, THE 
REST OF THE PIG IS USELESS.
BUT IF YOU HUMANIZE THE ENTIRE 
PIG, THEN YOU CAN GET TEN 
DIFFERENT, SOMETIMES MORE TRANS
TRANSPLANTS.
ALL THE SAME TISSUES YOU WILL 
GET FROM A HUMAN, YOU CAN GET 
FROM A PIG.
>>  OVER HERE.
>>  HELLO.
THE FIRST IS A VERY SMALL 
QUESTION.
YOU SHOWED HE HAD -- YOU SHOW HE
HEED AT THE BEGINNING THE 
ABUNDANCE OF THE CODONS IN THE 
GENOME.
COULD YOU TALK ABOUT HOW THAT 
RELATE TO THE ABUNDANCE OF THE 
DIFFERENT T /R-FPLS /TPHRNAS?
AND SORRY AND THAT RELATIVE 
ABUNDANCE IN DIFFERENT CELLS OR 
TISSUES?
>>  RIGHT.
SO THIS CODON TABLE IS VERY COOL
COOL.
I KNOW THERE ARE CELL TYPES AND 
IT DOES VAR FROM -- VARY FROM 
STATION AARY PHASE TO RAPIDLY 
REPRESELICATING.
FOR THE MOST PART THE NUMBER OF 
TRNAS CORRESPONDS TO THE 
ABUNDANCE IN /THIS TABLE.
THE EXCEPTION IS METHIANNINE, 
WHICH IS ESPECIALSPECIAL.
BUT THERE IS A CORRELATION.
AND SO WHEN WE RECODE, IF WE 
WANT /TO CHANGE IT FROM ONE SIN
SYNONYM TO ANOTHER, THEN WE NEED
TO INCREASE THE NUMBER OF TRNAS 
OR RELEASE FACTORS TO COMPENSATE
FOR THAT PROBABLY.
BUT THEY ARE DEFINITELY USED FOR
REGULATION.
BUT IT'S KIND OF -- IT'S A 
NUANCE SO WE CAN LAYER ON TOP OF
IT.
>>  AND IN DIFFERENT -- I ALSO 
HAVE A SECOND QUESTION.
IN DIFFERENT CELLS YOU FIND THE 
SAME EXPRESSION OF THE DIFFERENT
T /R-FPRNAS?
>>  NO.
T /R-FPRNAS ARE LEVELS DEPENDING
ON 
CELL TYPE.
THE SECOND QUESTION IS BROADER
BROADER.
HOW DO YOU SEE ALL THESE GENOMIC
THINGS CONVERTING INTO PERSONAL 
MEDICINE?
SO IN TERMS OF PUBLIC VERSUS 
PRIVATE RESEARCH AND HOW MUCH 
THESE THINGS COST AND HOW IT'S 
GOING TO DEVELOP?
>>  YEAH, THAT'S A PRETTY BIG 
QUESTION BUT I'D LOVE TO TALK TO
YOU OR A BUNCH OF YOU AFTERWARDS
AFTERWARDS.
BUT I THINK THAT WE HAVE A HUGE 
OPPORTUNITY HERE WITH THE 
TECHNOLOGIES STILL GOING EX
EXKNOWNST, WHERE I THINK WE'RE 
GOING TO VERY SOON HAVE? 
ADDITION TO CAMERAS ON YOUR 
PHONE, A SEQUENCER ON YOUR PHONE
PHONE, TOO, THAT THE NAN /OPORT 
TECHNOLOGY IS REALLY GOING INTO 
THAT DIRECTION OF PORTABILITY 
AND LOW COST SO YOU CAN ANALYZE 
YOUR ENVIRONMENT, WHICH IS 
SOMETHING HUGELY MISSING FROM 
THE STUDIES SO FAR.
>>  OVER HERE.
WE'LL TAKE TWO MORE QUESTIONS.
>>  HI, THANK YOU FOR THE TALK.
I HAVE A QUESTION REGARDING 
BUILDING THE ORGANIOIDS.
ONE OF THE PROJECTS IS BUILDING 
VAS /KHCULATURE INTO THE
ORGANIZS AND 
HOW THAT WAS A CHALLENGE.
MY QUESTION IS DO YOU THINK 
BUILDING THE ENTIRE, SAY, 
FULFILL FUNCTION /AAL BRAIN,
EACH 
OF THE SORT OF UNITS HAVE A RE
RE/KUCURSIVE PROGRAMMING,
MEANING 
THAT THEY SELF-GROW AND THEY 
SORT OF IN ORGANIZING TO /-THE 
BEAUTIFUL STRUCTURE OF THE BRAIN
BRAIN?
OR DO YOU THINK THAT AT SOME 
POINT THERE ARE DIRECTIVE OR 
PLAN THAT SORT OF SHAPES THE 
FINAL PRODUCT?
SO WHETHER THIS IS MORE 
INDIVIDUAL BLOCKS FORMED 
TOGETHER INTO COMPLEXITY OR MORE
A MASSIVE PLAN DIRECTING?
>>  RIGHT 
THE COURT:^ SO ANOTHER BIG 
QUESTION, BUT IT'S AN OPEN ONE.
AND IT'S EVEN MORE OPEN THAN IT 
MIGHT SEEM BECAUSE WE HAVE THE 
WAY IT NORMALLY HAPPENS AND THEN
WHAT SYNTHETIC BIOLOGY CAN DO 
WITH IT.
WE'VE BEEN PLEASANTLY SURPRISED 
NOW WITH OUR SYNTHETIC 
DEVELOPMENTAL BIOLOGY THAT WE 
CAN TAKE THINGS THAT NORMALLY 
TAKE HUNDREDS OF DAYS IN VIVO 
AND HUNDREDS OF DAYS IN MOST 
PROTOCOLS AND SHORTEN THEM TO 
FOUR DAYS.
IN FACT, FOUR DAYS IS KIND OF 
OUR STANDARD FOR MANY OF THESE 
PROTOCOLS.
WE NEED TO BE INDIVIDUVIGILANT
THAT WHAT
WE'RE GETTING IS EITHER NATURAL 
OR ACCEPTABLE.
AND IN PARTICULAR, MAKING A 
HUMAN ORGANI TAKES, AN
ADULT-SIZE
ORGANI TAKES DECADES.
ARE THERE SHORT CUTS, OR ARE WE 
GOING TO HAVE TO GO -- CLEARLY 
THE PIG DOES IT IN LESS TIME 
THAN WE DO.
AND THAT'S SOMETHING THAT'S 
QUITE LARGE.
SO IT'S A REALLY INTERESTING 
QUESTION.
CAN WE USE SYNTHETIC APPROACHES 
TO MAKING ORGANIS FASTER AND 
POSSIBLY BETTER?
BY BETTER, I MEAN REDUCED CANCER
CANCER, HE DEUCEED VIRAL AND RE
REDUCED SENESCENCE.
THAT'S SOMETHING THAT WILL BE 
HARD TO DO IN HUMANS BUT -- SO 
ESSENTIALLY THE APPROVAL 
PRESSURES ARE AGAINST IT IN 
HUMANS, BUT THEY WILL BE IN 
FAVOR OF IT WHEN YOU ARE DEALING
WITH ORGANIS.
SO I THINK THERE ARE SOME REALLY
INTERESTING OPPORTUNITIES.
>>  LAST QUESTION.
>>  YOU COVERED THE WORLD OF 
GENOMICS AND ALSO ULTIMATELY THE
HUMAN FRONTEERS OF BRAIN.
SO YOU THINK SOMEDAY WE SHOULD 
BE ABLE TO IDENTIFY CLUSTERS OF 
GENOME FOR HIGHER INTELLECTUAL 
ABILITY OF COG /TPHENITIVE
PROCESSING 
AND ALL THE OTHER NUR /O*EURONAL
FUNCTIONS?
>>  WELL, THERE IS TWO SHOULDS 
HERE.
ONE IS TECHNICALLY AND ANOTHER 
IS SOCIETALLY.
MY GUESS IS THAT BOTH WILL 
HAPPEN.
AND PART OF IT IS THERE IS GOING
TO BE HIGH MOTIVATION TO DEAL 
WITH ALZHEIMER'S AND OTHER
NEWUEURO
NEURODE/SKWRERPGENERIC DISEASES
AND SOME
OF THOSE SOLUTIONS WILL BE 
EFFECT /KWREUIVE IN PEOPLE WHO
ARE NOT 
ALREADY DE/SKWRERPGENERATING.
THEY WILL BE ENHANCING.
IN THE SAME SENSE THE VACCINES 
ARE ENHANCING.
THEY MAKE US SOUPER IMMUNE TO A 
VARIETY OF PATH GENS.
PATHOGENS.
AND WE'VE ALREADY SEEN IN MICE 
THAT IF YOU HAVE A SPECIFIC SET 
OF COG /TPHE!NITIVE TASKS IN
MIND, YOU 
CAN, WITH ONE OR TWO GENES, MAKE
A HUGE CHANGE.
SO I DON'T THINK IT'S GOING TO 
BE FUNDAMENTALLY DIFFERENT FROM 
OTHER THINGS.
AND WE SHOULDN'T USE NATURAL 
VARIATION OF POPULATION AS OUR
OURONOUR 
ONLY GUIDE POST BECAUSE THERE 
ARE SYNTHETIC THINGS YOU CAN DO.
MOST OF FAPHARMACOLOGY IS NOT 
NATURAL AND YOU END UP WITH 
THINGS THAT ARE IN SOME CASES 
FAR BEYOND THE NATIVE STATE
/-OFIB
HUMAN BEINGS.
BUT WE NEED TO THINK ABOUT THIS 
VERY CAREFULLY AND TALK ABOUT IT
IN ADVANCE, AS WE ARE DOING 
TODAY.
>>  CONGRATULATIONS, THANK YOU.
>>  SO WHAT A WONDERFUL ROMP 
WE'VE HAD THROUGH TECHNOLOGY, 
/SREUGSZ OF THE FUTURE, ETHICS 
THAT HAVE TO BE VERY MUCH IN
IN/KRORCORPORATED INTO ALL OF
THIS, 
WHICH GEORGE HAS EMPHASIZED 
THROUGHOUT ALL OF THESE 
ADVENTURES.
I THINK IF MARSHAL NUREMBERG HAD
BEEN HERE, HE WOULD HAVE LOVED 
THIS.
WE CAN JOIN NOW IN THE LIBRARY 
FOR SOME /SKPOEUF COOKIES, BUT 
-- COFFEE AND COOKIES, BUT 
PLEASE, LET'S THANK GEORGE AGAIN
AGAIN.
/PHRA
[APPLAUSE]
