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张锋团队重新设计了Cas12b，带来第三个基因组编辑系统 |
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论文标题：Engineering of CRISPR-Cas12b for human genome editing
期刊：
作者：Jonathan Strecker,Sara Jones, Balwina Koopal, Jonathan Schmid-Burgk, Bernd Zetsche, Linyi Gao, Kira S. Makarova, Eugene V. Koonin, Feng Zhang
发表时间：2019/01/22
数字识别码： s41467-018-08224-4
原文链接：
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《自然-通讯》本周发表的一篇论文Engineering of CRISPR-Cas12b for human genome editing手机版了第三个可以编辑人类细胞基因组的CRISPR-Cas系统。
图1 嗜高温核酸酶Cas12b的鉴定。图源：Strecker等
CRISPR-Cas9是一个多功能基因组编辑系统，但Cas9并非Cas蛋白家族中唯一一种RNA导向的核酸酶（即一种能切割DNA的酶）。除了Cas9之外，研究人员还发现了Cas12a和Cas12b。Cas12a已被开发成基因组编辑工具，而Cas12b尚未被完全开发，这其中至少有一部分原因是它嗜高温的特性。
美国麻省理工学院-哈佛大学博德研究所的张锋和同事对Cas12b进行了研究，因为这种蛋白比Cas9或Cas12a更小，更容易通过病毒载体实现细胞间递送。但原始结构的Cas12b会切割双链DNA中的非靶标单链。为了解决这一问题，作者对Cas12b重新进行了设计，增强其在人体体温（37°C）下的活性。与Cas9相比，重新设计的Cas12b在细胞培养实验中对靶序列具有更高的特异性。
图2 BhCas12b v4是一种高度特异性的核酸酶。图源：Strecker等
想要将Cas12b改造成和Cas9一样应用广泛的工具，还有很多工作要做，但第三个潜在基因组编辑系统的出现会给研究人员更多选择
摘要：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.
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