CPF1

Cpf1IsaSingleRNA-GuidedEndonucleaseofaClass2CRISPR-CasSystemBackgroundNaturalMechanismsofMicrobialCRISPRSystemsinAdaptiveImmunity.SaCas9ismorethan1kilobaseshorterSaCas9caneditthegenomewithefficienciessimilartothoseofSpCas9HighlightsCRISPR-Cpf1isaclass2CRISPRsystem.Cpf1isaCRISPR-associatedtwo-componentRNAprogrammableDNAnuclease.TargetedDNAiscleavedasa5-ntstaggeredcutdistaltoa5’T-richPAM.TwoCpf1orthologsexhibitrobustnucleaseactivityinhumancells.Cas9CRISPR1.0CRISPR2.0VSOrganizationoftwoCRISPRlocifoundinFrancisellanovicidaU112(NC_008601).ThedomainarchitecturesofFnCas9andFnCpf1arecompared.Cpf1-ContainingCRISPRLociAreActiveBacterialImmuneSystemsTypeVCpf1-ContainingCRISPRLociAreActiveBacterialImmuneSystemsSchematicillustratingtheplasmiddepletionassayfordiscoveringthePAMpositionandidentity.ThePAMforFnCpf1islocatedupstreamofthe5'endofthedisplacedstrandoftheprotospacerandhasthesequence5'-TTN.ThemiddleTismorecriticalforPAMrecognitionthanthefirstTandthat,inagreementwiththesequencemotifsdepletedinthePAMdiscoveryassay,thePAMmightbemorerelaxedthan5'-TTNCpf1-ContainingCRISPRLociAreActiveBacterialImmuneSystemsTheCpf1-AssociatedCRISPRArrayIsProcessedIndependentofTracrRNASmallRNA-seqofFrancisellanovicidaU112.FnCpf1anditsCRISPRarrayaretheonlyelementsrequiredfromtheFnCpf1locustoachievecrRNAprocessing.TheCpf1-AssociatedCRISPRArrayIsProcessedIndependentofTracrRNABTheCpf1-AssociatedCRISPRArrayIsProcessedIndependentofTracrRNAE.coliharboringdifferenttruncationsoftheFnCpf1CRISPRlocusshowsthatonlyFnCpf1andtheCRISPRarrayarerequiredforplasmidDNAinterferenceCpf1IsaSinglecrRNA-GuidedEndonucleaseFnCpf1alongwithanin-vitro-transcribedmaturecrRNA-targetingprotospacer1wasabletoefficientlycleavethetargetplasmidinaMg2+-andcrRNA-dependentmanner.Moreover,FnCpf1wasabletocleavebothsupercoiledandlineartargetDNA.SangersequencingofthecleavedDNAends.WefoundthatFnCpf1mediatedcleavageresultsina5-nt5‘overhang.Cpf1IsaSinglecrRNA-GuidedEndonucleaseFnCpf1requiresthe5‘-TTNPAMtobeinaduplexforminordertocleavethetargetDNAandcanonlyrecognizethePAMincorrectlyWatson-CrickpairedDNA.SequenceandStructuralRequirementsfortheCpf1crRNAFnCpf1recognizesthecrRNAthroughacombinationofsequencespecificandstructuralfeaturesofthestemloop.SequenceandStructuralRequirementsfortheCpf1crRNATheRuvC-likeDomainofCpf1MediatesRNA-GuidedDNACleavageBATheRuvC-likedomainofFnCpf1cleavesbothstrandsofthetargetDNA,perhapsinadimericconfiguration.DcTheRuvC-likeDomainofCpf1MediatesRNA-GuidedDNACleavageCpf1-FamilyProteinsfromDiverseBacteriaShareCommoncrRNAStructuresandPAMsPhylogenetictreeof16Cpf1orthologsselectedforfunctionalanalysis.Cpf1-FamilyProteinsfromDiverseBacteriaShareCommoncrRNAStructuresandPAMsCpf1-FamilyProteinsfromDiverseBacteriaShareCommoncrRNAStructuresandPAMsEFCpf1-FamilyProteinsfromDiverseBacteriaShareCommoncrRNAStructuresandPAMsCpf1CanBeHarnessedtoFacilitateGenomeEditinginHumanCellsCpf1CanBeHarnessedtoFacilitateGenomeEditinginHumanCellsDiscussionCpf1isasinglecrRNA-guidedendonuclease.Cpf1generatesastaggeredcutwitha5’overhang.Cpf1mightaidtheintroductionofnewDNAsequences.TheT-richPAMsoftheCpf1-familyalsoallowforapplicationsingenomeeditinginorganismswithparticularlyAT-richgenomes..Thanksforyourattention!