CRISPR-Cas12b 工程在人类基因组编辑中的应用。
Engineering of CRISPR-Cas12b for human genome editing.
机构信息
Howard Hughes Medical Institute, Cambridge, USA.
Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA.
出版信息
Nat Commun. 2019 Jan 22;10(1):212. doi: 10.1038/s41467-018-08224-4.
The type-V CRISPR effector Cas12b (formerly known as C2c1) has been challenging to develop for genome editing in human cells, at least in part due to the high temperature requirement of the characterized family members. Here we explore the diversity of the Cas12b family and identify a promising candidate for human gene editing from Bacillus hisashii, BhCas12b. However, at 37 °C, wild-type BhCas12b preferentially nicks the non-target DNA strand instead of forming a double strand break, leading to lower editing efficiency. Using a combination of approaches, we identify gain-of-function mutations for BhCas12b that overcome this limitation. Mutant BhCas12b facilitates robust genome editing in human cell lines and ex vivo in primary human T cells, and exhibits greater specificity compared to S. pyogenes Cas9. This work establishes a third RNA-guided nuclease platform, in addition to Cas9 and Cpf1/Cas12a, for genome editing in human cells.
V 型 CRISPR 效应物 Cas12b（以前称为 C2c1）在人类细胞中的基因组编辑方面一直难以开发，这至少部分是由于特征家族成员的高温要求。在这里，我们探索了 Cas12b 家族的多样性，并从枯草芽孢杆菌中鉴定出一种有前途的人类基因编辑候选物，即 BhCas12b。然而，在 37°C 时，野生型 BhCas12b 优先在非靶标 DNA 链上进行缺口切割，而不是形成双链断裂，导致编辑效率降低。我们使用多种方法鉴定出了克服这一限制的 BhCas12b 的功能获得性突变。突变型 BhCas12b 促进了人类细胞系和原代人 T 细胞中的强大基因组编辑，并且与 S. pyogenes Cas9 相比具有更高的特异性。这项工作建立了除 Cas9 和 Cpf1/Cas12a 之外的第三个用于人类细胞基因组编辑的 RNA 引导核酸酶平台。
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