FGTB Next-Generaon Technology Bootcamp at ATVB|PVD …...FGTB Next-Generaon Technology Bootcamp at...
12
FGTB Next-Genera.on Technology Bootcamp at ATVB|PVD 2017 Base edi.ng as an alterna.ve to genome edi.ng Kiran Musunuru, MD, PhD, MPH, FAHA FINANCIAL DISCLOSURES: None UNLABELED/UNAPPROVED USES DISCLOSURES: None
Transcript of FGTB Next-Generaon Technology Bootcamp at ATVB|PVD …...FGTB Next-Generaon Technology Bootcamp at...
-
FGTBNext-Genera.onTechnologyBootcampatATVB|PVD2017
Baseedi.ngasanalterna.vetogenomeedi.ng
KiranMusunuru,MD,PhD,MPH,FAHA
FINANCIALDISCLOSURES:NoneUNLABELED/UNAPPROVEDUSESDISCLOSURES:None
-
Baseedi.ng
•Genomeedi.ngfacilitatesknockoutorknock-inofmuta.onsbyintroducingadouble-strandbreakatadesiredsiteingenome
•Baseedi.ngintroducessequence-specificmuta.ons–withouttheneedfordouble-strandbreaks,andmuchmoreefficientlythanstandardgenomeedi.ng
•Canbeusedinvitroandinvivo
-
Baseedi.ng
CanspecificallyintroduceC!TandG!Achangeswithoutdouble-strandDNAbreaks,withdecreasedoff-targeteffects
-
CCGACTACGTCCTGGAGTGTUAUUACGTAATGCACGCGGTGATTACCACCCCTAGCAAGG GGCTGATGCAGGACCTCACAGTGGTGCATTACG TGCGCCACTAATGGTGGGGATCGTTCC
CCGACTACGTCCTGGAGTGTUAUUACGTAATGCACGCGGTGATTACCACCCCTAGCAAGG GGCTGATGCAGGACCTCACAATAATGCATTACGTGCGCCACTAATGGTGGGGATCGTTCC * **
deamina.onofC’stoU’s,baseexcisionrepairisblocked
nickrepairreplacementofU-GbasepairswithU-Abasepairs
CCGACTACGTGGTCCAGTGTCACCACGTAATGCACGCGGTGATTACCACCCCTAGCAAGGGGCTGATGCACCAGGT GCCACTAATGGTGGGGATCGTTCC \ / CACAGTGGTGCATTACGTGC
20-bpprotospacer
GUGUCACCACGUAAUGCACG
Cas9nickase
gRNA
APOBEC1cytosinedeaminase
uracilDNAglycosylaseinhibitor(blocksrepairofdeamina.on)
BE3baseeditor
“BE3”
-
Targe.ngasiteinthegenome
PickingatargetsitewithBE3:
1. DesignaguideRNAinthesamewayyouwouldforstandardCRISPR-Cas9(needsanNGGPAM,shownhereinblue)
2. Deamina.onofcytosinesoccursonlyonthe“top”strand;ifyouwishtochangeguaninestoadenines,youneedtotargetthecytosinesontheotherstrand,whichmeansthePAMneedstobeontheotherstrand(i.e.,flipBE3upsidedown)
3. Deamina.onofcytosinescanoccuranywhereina“window”13to17nucleo.desupstreamofthePAM(shownhereasthebasesoverlappingthedeaminasedomain)
4. Ofenallofthecytosinesinthewindowwillbedeaminatedandeditedintothymines–youneedtoaccountforthisinyourexperimentaldesign!!
CCGACTACGTGGTCCAGTGTCACCACGTAATGCACGCGGTGATTACCACCCCTAGCAAGGGGCTGATGCACCAGGT GCCACTAATGGTGGGGATCGTTCC \ / CACAGTGGTGCATTACGTGC
20-bpprotospacer
GUGUCACCACGUAAUGCACG
Cas9nickase
gRNA
APOBEC1cytosinedeaminase
uracilDNAglycosylaseinhibitor(blocksrepairofdeamina.on)
BE3baseeditor
-
Usingbaseeditortointroducenonsensemuta.ons
PAMisinbluewindowisinpink
-
Usingbaseeditortointroducenonsensemuta.ons
PAMisinbluewindowisinpink
Watchout:targe.ngisontheoppositestrand!!
-
PCSK9wild-type
PCSK9Q278X(CAG!TAG)
12
1=control2=BE3
baseeditor3(BE3)inHEK293Tcells
BaseeditorintroducesPCSK9nonsensemuta.on
-
Off-targeteffects
•Althoughbaseeditorsdonotrequiredouble-strandbreaks,some.mesindelsdooccur(sincetheoppositestrandisnickedbyCas9)
•Genome-wideprofilingofBE3vs.standardCas9hasshownthatBE3hasfeweroff-targetsites,withmostoftheoff-targeteffectsbeingbaseedits(ratherthantheindelsobservedwithstandardCas9)
•Baseedi.ngseemssaferthangenomeedi.ng
-
•Iftryingtoeditapar.cularcytosinebasetothymine,seeifthereisanappropriatePAM(NGG)13to17basesdownstreamofthecytosine
•Iftryingtoeditapar.cularguaninebasetoadenine,seeifthereisanappropriatePAM(CCN)13to17basesupstreamofthecytosine(sinceyouareactuallytarge.ngtheoppositestrand)
Tipsforiden.fyingtargetsite
-
ANGPTL3codingsequence(fromgenomesequence)
ATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGATCAAGACAATTCATCATTTGATTCTCTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATG...
Example
D22Nmuta.on=GAC!AAC
Sinceedi.ngaG,lookfor“CCN”13to17bpupstream
Asingle“CCN”(PAMonoppositestrand)placesGin“window”(15bpawayfromPAM)
-
ANGPTL3codingsequence(fromgenomesequence)
ATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGATCAAGACAATTCATCATTTGATTCTCTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATG...
Example
protospacer=ATTGTCTTGATCAATTCTGG
G!Aeditwilloccur15bpawayfromthePAM
SincetherearenootherG’swithinthewindow,onlythedesiredGwillbeeditedtoA
