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改造的CRISPR-Cas9变体实现无限制的基因组靶向
作者:小柯机器人 发布时间:2020/3/28 21:49:39

美国马萨诸塞州总医院Benjamin P. Kleinstiver研究组利用工程化改造的CRISPR-Cas9变体-近PAMless,实现无限制的基因组靶向。2020年3月26日出版的《科学》在线发表了这项成果。

为了消除靶位点识别的限制,研究人员改造了化脓链球菌Cas9(SpCas9)的变体,以消除NGG 原间隔子相邻基序(PAM)的要求。研究人员设计了一种名为SpG的变体,能够靶向一组扩展的NGN PAM;并进一步优化了该酶,产生了一种近PAMless SpCas9变体,名为SpRY(NRN> NYN PAM)。 SpRY核酸酶和碱基编辑器变体可以靶向几乎所有PAM,并且在人类细胞中对具有NRN PAM的广泛位点均表现出强大的活性,而对于具有NYN PAM的酶则具有较低但重要的活性。

使用SpG和SpRY,研究人员生成了以前无法获得的与疾病相关的遗传变异,从而实现在基因组编辑应用中进行高分辨率的靶向。

据悉,CRISPR-Cas酶对DNA的编辑需要对PAM识别,从而将靶位点识别限制在序列子集中。

附:英文原文

Title: Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants

Author: Russell T. Walton, Kathleen A. Christie, Madelynn N. Whittaker, Benjamin P. Kleinstiver

Issue&Volume: 2020/03/26

Abstract: AbstractManipulation of DNA by CRISPR-Cas enzymes requires the recognition of a protospacer adjacent motif (PAM), limiting target site recognition to a subset of sequences. To remove this constraint, we engineered variants of Streptococcus pyogenes Cas9 (SpCas9) to eliminate the NGG PAM requirement. We developed a variant named SpG capable of targeting an expanded set of NGN PAMs, and further optimized this enzyme to develop a near-PAMless SpCas9 variant named SpRY (NRN>NYN PAMs). SpRY nuclease and base-editor variants can target almost all PAMs, exhibiting robust activities on a wide range of sites with NRN PAMs in human cells and lower but substantial activity on those with NYN PAMs. Using SpG and SpRY, we generated previously inaccessible disease-relevant genetic variants, supporting the utility of high-resolution targeting across genome editing applications.

DOI: 10.1126/science.aba8853

Source: https://science.sciencemag.org/content/early/2020/03/25/science.aba8853

期刊信息
Science:《科学》,创刊于1880年。隶属于美国科学促进会,最新IF:41.037