缩短 sgRNA-DNA 界面使 SpCas9 和 eSpCas9(1.1)能够切割靶 DNA 链。

Shortening the sgRNA-DNA interface enables SpCas9 and eSpCas9(1.1) to nick the target DNA strand.

机构信息

Key Laboratory of Agricultural Water Resources, Hebei Laboratory of Agricultural Water-Saving, Center for Agricultural Resources Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Shijiazhuang, 050022, China.

State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, 100101, China.

出版信息

Sci China Life Sci. 2020 Nov;63(11):1619-1630. doi: 10.1007/s11427-020-1722-0. Epub 2020 Jun 24.

DOI:10.1007/s11427-020-1722-0

PMID:32592086

Abstract

The length of the sgRNA-DNA complementary sequence is a key factor influencing the cleavage activity of Streptococcus pyogenes Cas9 (SpCas9) and its variants. The detailed mechanism remains unknown. Here, based on in vitro cleavage assays and base editing analysis, we demonstrate that reducing the length of this complementary region can confer nickase activity on SpCas9 and eSpCas9(1.1). We also show that these nicks are made on the target DNA strand. These properties encouraged us to develop a dual-functional system that simultaneously carries out double-strand DNA cleavage and C-to-T base conversions at separate targets. This system provides a novel tool for achieving trait stacking in plants.

摘要

sgRNA-DNA 互补序列的长度是影响化脓链球菌 Cas9(SpCas9)及其变体切割活性的关键因素。其详细机制尚不清楚。在这里，我们通过体外切割实验和碱基编辑分析表明，缩短该互补区域的长度可以使 SpCas9 和 eSpCas9(1.1)具有切口酶活性。我们还表明，这些切口是在靶 DNA 链上产生的。这些特性促使我们开发了一种双功能系统，该系统可以在分离的靶标上同时进行双链 DNA 切割和 C 到 T 的碱基转换。该系统为植物实现性状叠加提供了一种新的工具。