分子生物学读书报告CRISPR-Cas9..pptVIP

结合后的示意图TargetsequenceheteroduplexformswhengRNAbindstothetargetsequenceviaWatsoneCrickbasepairing.inthesamemanner,therepeatandanti-repeatregionsformtherepeat:anti-repeatduplex,whichisessentialforCas9function.Concurrently,theremainingtracrRNAbasesformstemloops1,2,and3viathree,four,andsixWatsoneCrickbasepairs,respectively.Throughtheaboveinteractions,aCas9-sgRNAbinarycomplexisformed.以产脓链球菌为例先后形成复合物然后对外源DNA进行干扰。切开基因后有两种不同的修复方式：TheNHEJpathwayinducesunpredictableinsertions,deletions,orsubstitutionswiththeefficiencyof20%e60%.TheHDRpathwayproducesprecisebasemutations,insertionsordeletionsthroughtheemploymentofhomologousdonorDNAsequenceswithalowfrequency.Thechoiceofpathwayisgovernedbythephasesofcellcycle,inwhichNHEJisrestrictedtoG1,SandG2phases,whileHDRprefersSandG2phases.转基因人类疾病的小鼠模型特定组织基因编辑同时生成多个基因突变灵活操作表观基因组新奇的潜力在RNA编辑方面图：编辑方面，可以删除、添加、激活或抑制其他生物体的目标基因，这些目标基因包括人、老鼠、斑马鱼、细菌、果蝇、酵母、线虫和农作物细胞内的基因。哺乳动物细胞中gRNA(chemricRNA)介导Cas9基因定点编辑诱导精准的HDR或NHEJ途径以及实现多路传输方法该领域迫切需要从总体上评估Cas9核酸酶的脱靶效应等。针对每一种细胞类型需要去优化等对于转换平衡远离NHEJ介导的插入突变HDR驱动途径的改变仍然是一个发展的当务之急。由于对其机理知之甚少，很可能或导致引发整个物种的变化的结果,可能对生态平衡产生影响。所以反对使用这种技术的言论逐渐上升。担忧采用CRISPR/Cas9系统后产生的伦理问题，就像克隆一样所以有些国家已经禁止在人类身上使用该技术。CRISPR/Cas9systemGenomeeditingtechnologies,includingchemical-andUV-inducedmutagenesis,DNArecombinase-mediatedgenereplacement,zinc-fingernucleases(ZFNs),andtranscriptionalactivator-likeeffectornuclease(TALEN)systems,haveprofoundlycontributedtobothfundamentalandclinicaladvancesinbiologicalresearch(Urnovetal.,2005;Bedelletal.,2012).Recently,theCRISPR/Cassystem,derivedfromtheadaptiveimmunesystemofthebacteriumStreptococcuspyogeneshasdramaticallyincreasedourabilitytoeditthegenomesofdiversespecies(Sapranauskasetal.,2011).GENOMEEDITINCONTENTTodate,threetypesofCRISPR/Cassystems(I,II,andIII)haveb