Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells.
Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells.
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DOI：
10.1038/nbt.3620
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发表时间：
2016-08
影响因子：
46.9
通讯作者：
Joung JK
中科院分区：
文献类型：
--
作者：
Kleinstiver BP;Tsai SQ;Prew MS;Nguyen NT;Welch MM;Lopez JM;McCaw ZR;Aryee MJ;Joung JK
CRISPR-Cas Cpf 1核酸酶的活性和全基因组特异性尚未明确。我们表明，来自氨基酸球菌BV 3L 6和毛螺菌科细菌ND 2006的两种Cpf 1核酸酶（分别为AsCpf 1和LbCpf 1）在人类细胞中具有与广泛使用的化脓性链球菌Cas9（SpCas 9）相当的靶向效率。我们还报告说，用于编程Cpf 1核酸酶的短CRISPR RNA（crRNA）的3'端的4到6个碱基对单碱基错配不敏感，但crRNA该区域的许多其他碱基对单或双取代高度敏感。使用两种Cpf 1核酸酶进行的GUIDE-seq和靶向深度测序分析，我们无法检测到20种不同crRNA中超过一半的脱靶切割。我们的研究结果表明，AsCpf 1和LbCpf 1在人类细胞中具有高度特异性。
The activities and genome-wide specificities of CRISPR-Cas Cpf1 nucleases are not well defined. We show that two Cpf1 nucleases from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) have on-target efficiencies in human cells comparable with those of the widely used Streptococcus pyogenes Cas9 (SpCas9). We also report that four to six bases at the 3’ end of the short CRISPR RNA (crRNA) used to program Cpf1 nucleases are insensitive to single base mismatches, but that many of the other bases in this region of the crRNA are highly sensitive to single or double substitutions. Using GUIDE-seq and targeted deep sequencing analyses performed with both Cpf1 nucleases, we were unable to detect off-target cleavage for more than half of 20 different crRNAs. Our results suggest that AsCpf1 and LbCpf1 are highly specific in human cells.