CRISPR-Cas12a-Assisted Recombineering in Bacteria.
CRISPR-Cas12a-Assisted Recombineering in Bacteria.
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CRISPR-Cas12a 辅助细菌重组工程
DOI：
10.1128/aem.00947-17
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发表时间：
2017-09-01
影响因子：
4.4
通讯作者：
Sun YC
中科院分区：
文献类型：
--
作者：
Yan MY;Yan HQ;Ren GX;Zhao JP;Guo XP;Sun YC
摘要簇状规则间隔短回文重复序列(CRISPR)-Cas12a(Cpf1)在许多生物中已成为一种有效的基因组编辑工具。在这里，我们开发和优化了CRISPR-Cas12a辅助的重组工程系统，以促进细菌的遗传操作。使用该系统，可以很容易地在大肠杆菌、鼠疫耶尔森菌和污垢分枝杆菌的染色体或天然质粒上产生点突变、缺失、插入和基因替换。由于CRISPR-Cas12a辅助重组工程不需要在染色体中引入抗生素耐药基因来选择重组子，因此它是在细菌中产生无标记和无疤痕突变的有效方法。重要性CRISPR-Cas9系统已被广泛用于促进许多细菌的基因组编辑。CRISPR-Cas12a(Cpf1)是一种新型的CRISPR-CAS系统，当与重组工程相结合时，它可以在细菌中进行高效的基因组编辑。Cas12a和Cas9识别不同的靶点，这使得更精确地选择切割靶点和引入所需的突变。此外，CRISPR-Cas12a辅助的重组工程还可用于质粒的遗传操作和质粒治愈。最后，系统分析了Cas12a辅助的重组工程在细菌中产生点突变、缺失、插入和替换的过程。综上所述，我们的发现将指导高效的Cas12a介导的细菌基因组编辑。
ABSTRACT Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a (Cpf1) has emerged as an effective genome editing tool in many organisms. Here, we developed and optimized a CRISPR-Cas12a-assisted recombineering system to facilitate genetic manipulation in bacteria. Using this system, point mutations, deletions, insertions, and gene replacements can be easily generated on the chromosome or native plasmids in Escherichia coli, Yersinia pestis, and Mycobacterium smegmatis. Because CRISPR-Cas12a-assisted recombineering does not require introduction of an antibiotic resistance gene into the chromosome to select for recombinants, it is an efficient approach for generating markerless and scarless mutations in bacteria. IMPORTANCE The CRISPR-Cas9 system has been widely used to facilitate genome editing in many bacteria. CRISPR-Cas12a (Cpf1), a new type of CRISPR-Cas system, allows efficient genome editing in bacteria when combined with recombineering. Cas12a and Cas9 recognize different target sites, which allows for more precise selection of the cleavage target and introduction of the desired mutation. In addition, CRISPR-Cas12a-assisted recombineering can be used for genetic manipulation of plasmids and plasmid curing. Finally, Cas12a-assisted recombineering in the generation of point mutations, deletions, insertions, and replacements in bacteria has been systematically analyzed. Taken together, our findings will guide efficient Cas12a-mediated genome editing in bacteria.