Repurposing CRISPR-Cas12b for mammalian genome engineering
Repurposing CRISPR-Cas12b for mammalian genome engineering
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DOI：
10.1038/s41421-018-0069-3
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发表时间：
2018-11-27
期刊：
影响因子：
33.5
通讯作者：
Li, Wei
中科院分区：
文献类型：
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作者：
Teng, Fei;Cui, Tongtong;Li, Wei
原核CRISPR-Cas适应性免疫系统为开发基因组编辑工具提供了宝贵的资源，如CRISPR-Cas9和CRISPR-Cas 12 a/Cpf 1。最近，CRISPR-Cas 12 b/C2 c1，一种独特的V-B型系统，已被表征为双RNA引导的DNA内切核酸酶系统。虽然它在体外具有切割活性，但其对内源基因组的切割活性仍有待探索。此外，所报道的Cas 12 b直系同源物的最佳切割温度高于40 ° C，这不适合哺乳动物应用。在这里，我们报告了来自嗜酸脂环酸芽孢杆菌（AaCas 12 b）的Cas 12 b系统的鉴定，该系统在宽温度范围（31摄氏度-59摄氏度）内保持最佳核酸酶活性。AaCas 12 b可以被重新用于工程化哺乳动物基因组，用于多用途应用，包括单和多基因组编辑，基因激活和基因突变小鼠模型的生成。此外，全基因组测序揭示了AaCas 12 b介导的基因组编辑的高特异性和最小的脱靶效应。我们的发现将CRISPR-Cas 12 b确立为哺乳动物基因组工程的通用工具。
The prokaryotic CRISPR-Cas adaptive immune systems provide valuable resources to develop genome editing tools, such as CRISPR-Cas9 and CRISPR-Cas12a/Cpf1. Recently, CRISPR-Cas12b/C2c1, a distinct type V-B system, has been characterized as a dual-RNA-guided DNA endonuclease system. Though being active in vitro, its cleavage activity at endogenous genome remains to be explored. Furthermore, the optimal cleavage temperature of the reported Cas12b orthologs is higher than 40 degrees C, which is unsuitable for mammalian applications. Here, we report the identification of a Cas12b system from the Alicyclobacillus acidiphilus (AaCas12b), which maintains optimal nuclease activity over a wide temperature range (31 degrees C-59 degrees C). AaCas12b can be repurposed to engineer mammalian genomes for versatile applications, including single and multiplex genome editing, gene activation, and generation of gene mutant mouse models. Moreover, whole-genome sequencing reveals high specificity and minimal off-target effects of AaCas12b-meditated genome editing. Our findings establish CRISPR-Cas12b as a versatile tool for mammalian genome engineering.