CRISPR-Cas12a Nucleases Bind Flexible DNA Duplexes without RNA/DNA Complementarity
CRISPR-Cas12a Nucleases Bind Flexible DNA Duplexes without RNA/DNA Complementarity
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DOI：
10.1021/acsomega.9b01469
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发表时间：
2019-10-22
期刊：
影响因子：
4.1
通讯作者：
Qin, Peter Z.
中科院分区：
文献类型：
--
作者：
Jiang, Wei;Singh, Jaideep;Qin, Peter Z.
Cas12a（也称为“Cpf1”）是一种 2 类 V-A CRISPR 相关核酸酶，可以在特定位点切割双链 DNA。 Cas12a 效应酶由单个蛋白质和 CRISPR 编码的小 RNA (crRNA) 组成，已用于基因组编辑和操作。这里报告的工作检查了 Cas12a 效应酶和 DNA 双链体之间的体外相互作用，两条链之间的碱基配对状态不同。数据显示，在 crRNA 指导和 DNA 靶链之间不存在互补性的情况下，Cas12a 会与具有不配对片段的双链体结合。这些脱靶双链体结合在负责 RNA 引导的双链 DNA 结合的 Cas12a 位点，但由于缺乏 RNA/DNA 杂合体形成而未被切割。这种混杂的结合归因于原型间隔子相邻基序旁边存在的未配对片段诱导的 DNA 灵活性增加。结果表明 Cas12a 的靶点辨别可能受到 DNA 灵活性的影响。因此，在基于 Cas12a 的基因组工程应用中，除了线性序列外，还应考虑 DNA 的灵活性和其他物理特性。
Cas12a (also known as "Cpf1") is a class 2 type V-A CRISPR-associated nuclease that can cleave double-stranded DNA at specific sites. The Cas12a effector enzyme comprises a single protein and a CRISPR-encoded small RNA (crRNA) and has been used for genome editing and manipulation. Work reported here examined in vitro interactions between the Cas12a effector enzyme and DNA duplexes with varying states of base-pairing between the two strands. The data revealed that in the absence of complementarity between the crRNA guide and the DNA target-strand, Cas12a binds duplexes with unpaired segments. These off-target duplexes were bound at the Cas12a site responsible for RNA-guided double-stranded DNA binding but were not cleaved due to the lack of RNA/DNA hybrid formation. Such promiscuous binding was attributed to increased DNA flexibility induced by the unpaired segment present next to the protospacer-adjacent-motif. The results suggest that target discrimination of Cas12a can be influenced by flexibility of the DNA. As such, in addition to the linear sequence, flexibility and other physical properties of the DNA should be considered in Cas12a-based genome engineering applications.