1a) intro:enhancer/promoterregions(promoterinitiate/enhancerregulate) transcription2components:binding/effectordomains sgRNAtargetsDNA(targetedgene) dCas9nocleavagefunction,insteadfusedtoeffectordomainfortranscriptionregulation effectordomaincanbeactivatororrepressor

b)

sffvpromoter NLS:nuclearlocalizationsignal(transcriptionfactorsareinsidenucleus),tagsaprotein

tobeimportedinthenucleaus BFPtomeasure KRABrepressivedomain u6promoter dCas9KRABsgRNA

c)

designapplicattion 2strandsDNA,53codingstrand 8sgRNAs,5nottranslated(35)3translated GFPfluroescencequantifiedbyflowjoafter6daysoftransfection,normalizedtovector

control 3independenttrials sgRNAnecessary dCas9KRABfusionproteinmoreeffectiveindownregulation closertopromoterregion=moreeffective

d)

distributionofhistogramtellsgfpexpression negativecontrolsgRNAnotmuchrepression dCas9KRABplussgRNAtargettingGFPsuccessfulrepressionalmostequaltocontrol

withnoEGFPexpressionatalle)

nowactivationfunction insteadofKRAB,dcas9isbindedtoactivationdomain

twodCas9fusionproteinsconstructed targetingGAL4(studytranscription) transfectedcells,analyzed2dayslaterbyflowjoforGFPexpression dCas9VP64fusionandsgRNAmosteffective necessarycomponents

2a)

notunderstandmethodscompletely diagramhsowedthatminimalofftargetshighspecificity onlysgGFPsoGFPreducedsignal RNAcollected15daysafterlentiviraltransduction

2b)

similarfinding onlyGFPexpressionlevelisreduced therestofgenesisnormal data=2biologicalreplicates