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1、protocolCRISPRinterference(CRISPRi)forsequence-specificcontrolofgeneexpressionMatthewHLarson1–3,LukeAGilbert1–3,XiaowoWang4,WendellALim1–3,5,JonathanSWeissman1–3&LeiSQi1,3,51DepartmentofCellularandMolecularPharmacology,UniversityofCalifornia,SanFrancisco(UCSF
2、),SanFrancisco,California,USA.2HowardHughesMedicalInstitute,UCSF,SanFrancisco,California,USA.3CaliforniaInstituteforQuantitativeBiomedicalResearch,SanFrancisco,California,USA.4BioinformaticsDivision,CenterforSyntheticandSystemsBiology,TsinghuaNationalLaborato
3、ryforInformationScienceandTechnologyDepartmentofAutomation,TsinghuaUniversity,Beijing,China.5UCSFCenterforSystemsandSyntheticBiology,UCSF,SanFrancisco,California,USA.CorrespondenceshouldbeaddressedtoL.S.Q.(stanley.qi@ucsf.edu).Publishedonline17October2013;doi
4、:10.1038/nprot.2013.132sequence-specificcontrolofgeneexpressiononagenome-widescaleisanimportantapproachforunderstandinggenefunctionsandforengineeringgeneticregulatorysystems.Wehaverecentlydescribedanrna-basedmethod,crIsprinterference(crIspri),fortargetedsilen
5、cingoftranscriptioninbacteriaandhumancells.thecrIsprisystemisderivedfromtheStreptococcuspyogenescrIspr(clusteredregularlyinterspacedpalindromicrepeats)pathway,requiringonlythecoexpressionofacatalyticallyinactivecas9proteinandacustomizablesingleguiderna(sgrna)
6、.thecas9-sgrnacomplexbindstoDnaelementscomplementarytothesgrnaandcausesastericblockthathaltstranscriptelongationbyrnapolymerase,resultingintherepressionofthetargetgene.Hereweprovideaprotocolforthedesign,constructionandexpressionofcustomizedsgrnasfortranscript
7、ionalrepressionofanygeneofinterest.WealsoprovidedetailsfortestingtherepressionactivityofcrIspriusingquantitativefluorescenceassaysandnativeelongatingtranscriptsequencing.crIspriprovidesasimplifiedapproachforrapidgenerepressionwithin1–2weeks.themethodcanalsobe
8、adaptedforhigh-throughputinterrogationofgenome-widegenefunctionsandgeneticinteractions,thusprovidingacomplementaryapproachtornainterference,whichcanbeusedinawidervarietyoforganisms.IntroD
