Post on 04-Nov-2015
description
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