Enhancement of target specificity of CRISPR-Cas12a by using a chimeric DNA-RNA guide
Enhancement of target specificity of CRISPR-Cas12a by using a chimeric DNA-RNA guide
复制标题
DOI：
10.1093/nar/gkaa605
复制
发表时间：
2020-09-04
影响因子：
14.9
通讯作者：
Lee, Seung Hwan
中科院分区：
文献类型：
--
作者：
Kim, Hanseop;Lee, Wi-jae;Lee, Seung Hwan
CRISPR-Cas9系统被广泛用于靶向基因组工程。CRISPR- cas12a （Cpf1）是通过识别富含胸腺嘧啶的原间隔邻近基序（PAM）序列来控制靶基因的CRISPR效应物之一。与Cas9相比，Cas12a对向导RNA中的错配具有更高的敏感性；因此，脱靶序列的识别和切割较低。然而，它可以耐受原间隔器中远离PAM序列（TTTN或TTN）的区域的错配，当Cas12a活性出于治疗目的而得到改善时，脱靶切割问题可能会变得更加成问题。因此，我们研究了Cas12a的脱靶切割，并对Cas12a (cr)RNA进行了修饰，以解决脱靶切割问题。我们开发了一种CRISPR-Cas12a，它可以通过用DNA部分取代（cr）RNA来改变碱基对靶DNA的能量势，以高度特异性和有效的方式诱导靶DNA序列突变。提出了一个解释嵌合（cr）RNA引导CRISPR-Cas12a和SpCas9缺口酶如何在细胞内基因组中有效工作的模型。基于嵌合向导的CRISPR-Cas12a基因组编辑减少了脱靶切割，从而提高了安全性，这在治疗由基因突变引起的不治之症方面具有潜在的应用前景。
The CRISPR-Cas9 system is widely used for target-specific genome engineering. CRISPR-Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR-Cas12a that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR-Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR-Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.