小柯机器人

不受PAM序列限制的SpCas9
2020-02-13 10:09

化脓性链球菌Cas9(SpCas9)变体的连续进化与non-G 前间区序列邻近基序(PAMs)兼容。这一成果由博德研究所David R. Liu课题组取得,在线发表在2020年2月10日的国际学术期刊《自然—生物技术》上。

研究人员揭示了三种新SpCas9变体的进化历程,这些变体使用噬菌体辅助的非连续进化共同识别了NRNH PAM(其中R是A或G、H是A、C或T),三种新的噬菌体辅助连续进化策略首先结合DNA,第二选择是切割DNA。这些进化变体和SpCas9-NG的靶向能力在HEK293T细胞中进行了验证,该验证共使用了11776种基因组整合的前间区-sgRNA文库,其中包含所有可能的NNNN PAM。进化变体介导和形成了人类细胞中的插入缺失和碱基编辑,并且在之前无法使用CACC PAM的镰状贫血细胞中实现了A•T到G•C碱基的突变。这些新进化的SpCas9变体与以前报道的变体一起,原则上可以靶向大多数NR PAM序列,并大大降低了基于Cas9方法无法获得基因组位点的比例。

研究人员表示,SpCas9及其工程变体的靶向范围在很大程度上受限于含有G碱基的PAM序列。

附:英文原文

Title: Continuous evolution of SpCas9 variants compatible with non-G PAMs

Author: Shannon M. Miller, Tina Wang, Peyton B. Randolph, Mandana Arbab, Max W. Shen, Tony P. Huang, Zaneta Matuszek, Gregory A. Newby, Holly A. Rees, David R. Liu

Issue&Volume: 2020-02-10

Abstract: The targeting scope of Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants is largely restricted to protospacer-adjacent motif (PAM) sequences containing G bases. Here we report the evolution of three new SpCas9 variants that collectively recognize NRNH PAMs (where R is A or G and H is A, C or T) using phage-assisted non-continuous evolution, three new phage-assisted continuous evolution strategies for DNA binding and a secondary selection for DNA cleavage. The targeting capabilities of these evolved variants and SpCas9-NG were characterized in HEK293T cells using a library of 11,776 genomically integrated protospacer–sgRNA pairs containing all possible NNNN PAMs. The evolved variants mediated indel formation and base editing in human cells and enabled AT-to-GC base editing of a sickle cell anemia mutation using a previously inaccessible CACC PAM. These new evolved SpCas9 variants, together with previously reported variants, in principle enable targeting of most NR PAM sequences and substantially reduce the fraction of genomic sites that are inaccessible by Cas9-based methods.

DOI: 10.1038/s41587-020-0412-8

Source: https://www.nature.com/articles/s41587-020-0412-8

Nature Biotechnology:《自然—生物技术》,创刊于1996年。隶属于施普林格·自然出版集团,最新IF:68.164
官方网址:https://www.nature.com/nbt/
投稿链接:https://mts-nbt.nature.com/cgi-bin/main.plex


本期文章:《自然—生物技术》:Online/在线发表

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