High-frequency off-target mutagenesis induced by CRISPR-Cas 英文文献资料

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1、lettersHigh-frequencyoff-targetmutagenesisinducedbyCrIsPr-CasnucleasesinhumancellsYanfangFu1–4,JenniferAFoden1–3,CydKhayter1–3,MorganLMaeder1–3,5,DeepakReyon1–4,JKeithJoung1–5&JeffryDSander1–4Clustered,regularlyinterspaced,shortpalindromicrepeatTobegintodefinethedeterminantsofthespecificityofRGN

2、sin(CRISPR)RNA-guidednucleases(RGNs)haverapidlyemergedhumancells,wecarriedoutalarge-scaletestinwhichweassessedtheasafacileandefficientplatformforgenomeediting.Here,effectsofsystematicallymismatchingvariouspositionswithinmul-weuseahumancell–basedreporterassaytocharacterizetiplegRNA–targetDNAinter

3、faces.Todothis,weusedaquantitativeoff-targetcleavageofCRISPR-associated(Cas)9-basedRGNs.humancell–basedEGFPdisruptionassay,previouslydescribedbyourWefindthatsingleanddoublemismatchesaretoleratedlaboratory15,thatenablesrapidquantificationoftargetednucleasetovaryingdegreesdependingontheirpositiona

4、longtheactivities(Fig.1a).Inthisassay,theactivitiesofnucleasestargetedguideRNA(gRNA)-DNAinterface.WealsoreadilydetectedtoasingleintegratedEGFPreportergenecanbequantifiedbyassess-off-targetalterationsinducedbyfouroutofsixRGNstargetedinglossoffluorescencesignalinhumanU2OS.EGFPcellscausedbytoendoge

5、nouslociinhumancellsbyexaminationofpartiallyinactivatingframeshiftinsertion/deletion(indel)mutationsintro-mismatchedsites.Theoff-targetsitesweidentifiedharboredducedbyerrorpronenonhomologousend-joining(NHEJ)repairuptofivemismatchesandmanyweremutagenizedwithofnuclease-induceddouble-strandedbreaks

6、(Fig.1aandOnlinefrequenciescomparableto(orhigherthan)thoseobservedatMethods).Weusedthree~100-ntsinglegRNAs(sgRNAs)targetedtheintendedon-targetsite.OurworkdemonstratesthatRGNstodifferentsequenceswithinEGFP(SupplementaryFig.2);eachofcanbehighlyactiveevenwithimperfectlymatchedRNA-DNAthesesgRNAseffi

7、cientlydirectedCas9-mediateddisruptionofEGFPinterfacesinhumancells,afindingthatmightconfoundexpression(SupplementaryResults).theiruseinresearchandtherapeuticapplications.Ininitialexperiments,wetestedtheeffectsofsingle-nucleo