1、工程改造工程改造CRISPR-Cas9 复合体复合体：一种强有力的全基：一种强有力的全基因组功能基因筛选方法因组功能基因筛选方法An engineered CRISPR-Cas9 complex：a robust method for screening for functional gene in genome-scale OUTLINEMethods for elucidating gene functioncDNA library overexpression systemsGOFLOFGenome Edit ZFNTALENCRISPR/Cas9RNA interference 121
2、Genome EditCRISPR HistoryCRISPR Adaptive ImmunityCleavage StepResearchMedicine Gene therapyBiotechnologyGenome Engineering PlatformcDNA library overexpression systemsGOFLOFGenome Edit ZFNTALENCRISPR/Cas9RNA interference 121What do we need?Disadvantage:the complexity of transcript isoform variance cl
3、one into size-limited viral expression vectorscurrent dCas9activator :Activation efficiency Multi-sgRNA is not friendly to genome-wide GOFHow to Adhere?MS2?bacteriophage coat proteinsgrab other proteins p65、VP64、HSF1?activation domains recruits a distinct subset of transcription factors and chromati
4、n remodelling complexesWhich Module?12genes difficult to activate using dCas9VP64 and individual sgRNA1.0 guidesHow effective is SAM?For inter-gene variability：the basal expression level of each gene largely determines the level of activation.How to design sgRNA?For intragenic variability:targeting
5、within the-200 bp to+1 bp windowHow to design sgRNA?Multiplex gene activationSpecificity of SAM-mediated ActivationGenome-scale gene activation screenPrevious Research:enrichment of specific sgRNAs after PLX-4720 treatmentIdentification of top candidate genes using the RIGER P valueanalysis Research
6、 Result individual sgRNA and cDNA overexpression validationValidationengineered CRISPR-Cas9 complexa powerful genetic perturbation technology!Conclusion Silvana Konermann,Mark D.Brigham,Alexandro E.Trevino,Julia Joung,Omar O.AbudayyehClea Barcena,Patrick D.Hsu,Naomi Habib,Jonathan S.Gootenberg,Hiros
7、hi Nishimasu,Osamu Nureki&Feng Zhang.Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.Nature 14136(2014)Hsu,P.D.,Lander,E.S.&Zhang,F.Development and applications of CRISPR-Cas9 for genome engineering.Cell157,12621278(2014)Nishimasu,H.et al.Crystal structure of Cas9 in com
8、plex with guide RNA and targetDNA.Cell156,935949(2014)Shalem,O.et al.Genome-scale CRISPR-Cas9 knockout screening in human cells.Science343,8487(2014)ReferenceThanks for王金发、王宏斌老师一学期来的授课王金发、王宏斌老师一学期来的授课刘健恒同学与我的讨论刘健恒同学与我的讨论阅读大事记阅读大事记2015.5.1 Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex2015.6.13 Development and applications of CRISPR-Cas9 for genome engineering2015.6.14 Crystal structure of Cas9 in complex with guide RNA and targetDNA2015.6.15 Genome-scale CRISPR-Cas9 knockout screening in human cells
