1a)

b)

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

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
withnoEGFPexpressionatall

nowactivationfunction
insteadofKRAB,dcas9isbindedtoactivationdomain
twodCas9fusionproteinsconstructed
targetingGAL4(studytranscription)
transfectedcells,analyzed2dayslaterbyflowjoforGFPexpression
dCas9VP64fusionandsgRNAmosteffective
necessarycomponents

e)

2a)

2b)

notunderstandmethodscompletely
diagramhsowedthatminimalofftargetshighspecificity
onlysgGFPsoGFPreducedsignal
RNAcollected15daysafterlentiviraltransduction

similarfinding
onlyGFPexpressionlevelisreduced
therestofgenesisnormal
data=2biologicalreplicates