【摘要】 文库筛选技术广泛应用于生命科学研究各领域,加速了生物医药基础科研和临床实践的进展。本文对基于CRISPR-Cas9的文库类型和应用进行综述。CRISPR-Cas9文库包括敲除、活化和抑制文库。敲除文库通过Cas9/sgRNA靶向切割DNA序列,产生移码突变进行基因敲除。活化文库包括两种:一种是dCas9/sgRNA与转录活化蛋白质融合,例如dCas9-SAM,dCas9-SunTag和dCas9-VPR系统;另一种是dCas9与表观遗传修饰酶融合,例如d Cas9-Tet1和d Cas9-p300系统。CRISPR-Cas9抑制文库通过dCas9与表观遗传修饰蛋白质融合,抑制转录,例如d Cas9-KRAB和d Cas9-Dnmt3a系统。目前,CRISPR-Cas9文库广泛用于功能基因筛选、药物靶点和耐药靶点筛选、病毒靶点筛选和揭示信号通路,并在基因互作筛选及揭示顺式调节元件功能等方面初步展现其优势。CRISPR-Cas9文库优势在于其设计灵活、操作便捷、筛选高效。伴随基因编辑系统的研发,新的筛选文库靶向性和突变将更加精准,应用将更加拓展和深化。基于CRISPR-Cas9筛选文库不仅可以筛选病理和生理过程中的关键基因和非编码DNA,还可以揭示其发挥功能的分子机制,是剖析生命复杂调控网络的手术刀。更多还原

【Abstract】 The library screening technology is widely used in various fields of life science research,and it greatly accelerates the progress of basic research and clinical practice in biomedicine. This article reviews types and applications of libraries based on CRISPR-Cas9. CRISPR-Cas9 based libraries include knockout libraries,activation libraries,and inhibition libraries. The knockout library identifies and cuts specific DNA sequences through the Cas9/sgRNA complex,resulting in a frame shift mutation for gene knockout. Activation libraries include two types: one is dCas9/sgRNA fused to a transcriptional activator protein,such as dCas9-SAM,dCas9-SunTag and dCas9-VPR systems; the other is dCas9 fused to an epigenetic modification enzyme,such as dCas9-Tet1 and dCas9-p300 systems. Inhibitory libraries are fused to epigenetic modified proteins by dCas9 to inhibit transcription,such as d Cas9-KRAB and dCas9-Dnmt3 a systems. The advantages of the CRISPR-Cas9 library are its flexible design,easy operation and efficient screening. Currently,CRISPR-Cas9 based libraries play an irreplaceable role in functional gene screening,drug targets and resistance targets screening,viral targets screening,identification of signaling pathways,gene interaction screening,and revealing the function of cis-regulator elements. With the development of gene editing systems,novel screening libraries may emerge in the future,which target and mutate more precisely,and their applications will be deeper and expanded. CRISPR-Cas9 based libraries can not only screen key genes and non-coding DNAs in pathological and physiological processes,but also reveal the molecular mechanisms of their function. It is a scalpel that dissects the complex regulatory network of life.更多还原