RNA介导的人类基因组新工具

来源：仪方生物 www.yeslab.com

细菌和古细菌已经进化出被称之为规律成簇的间隔短回文重复（CRISPR）CRISPR关联（Cas）系统的适应性免疫防御系统，该系统利用短RNA介导外源核苷酸的降解。本项研究中，研究者构建2型细菌CRISPR系统来结合自设计的介导RNA（gRNA）在人类细胞中执行功能。对于胞内AAVS1位点，研究中获取到靶点的比率：在293T细胞10-25%、在K523细胞中13-8%、在诱导的多能干细胞中2-4%。这个过程依赖CRISPR组件，具有序列特异性，依赖多能gRNAs引发的刺激，能够影响多能靶位点的剪辑。研究者也计算了一个基因组范围的约190K特异gRNAs靶点为人类外显子的40.5%。通过本项研究，建立了一个易用、创新和多功能的RNA介导的基因组工程编辑工具。

RNA-Guided Human Genome Engineering via Cas9
Prashant Mali, Luhan Yang, Kevin M. Esvelt, John Aach, Marc Guell, James E. DiCarlo, Julie E. Norville, George M. Church

Bacteria and archaea have evolved adaptive immune defenses termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show this process relies on CRISPR components, is sequence-specific, and upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190k unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.