Laboratory of Experimental Virology, Department of Medical Microbiology, Amsterdam UMC, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
Department of Life and Environmental Sciences, University of Cagliari, Italy.
Nucleic Acids Res. 2020 Jun 4;48(10):5527-5539. doi: 10.1093/nar/gkaa226.
The CRISPR-Cas9 system has been used for genome editing of various organisms. We reported inhibition of the human immunodeficiency virus (HIV) in cell culture infections with a single guide RNA (gRNA) and subsequent viral escape, but complete inactivation of infectious HIV with certain combinations of two gRNAs. The new RNA-guided endonuclease system CRISPR-Cas12a (formerly Cpf1) may provide a more promising tool for genome engineering with increased activity and specificity. We compared Cas12a to the original Cas9 system for inactivation of the integrated HIV DNA genome. Superior antiviral activity is reported for Cas12a, which can achieve full HIV inactivation with only a single gRNA (called crRNA). We propose that the different architecture of Cas9 versus Cas12a endonuclease explains this effect. We also disclose that DNA cleavage by the Cas12a endonuclease and subsequent DNA repair causes mutations with a sequence profile that is distinct from that of Cas9. Both CRISPR systems can induce the typical small deletions around the site of DNA cleavage and subsequent repair, but Cas12a does not induce the pure DNA insertions that are routinely observed for Cas9. Although these typical signatures are apparent in many literature studies, this is the first report that documents these striking differences.
CRISPR-Cas9 系统已被用于各种生物体的基因组编辑。我们曾报道过，在细胞培养感染中，单条向导 RNA（gRNA）可抑制人类免疫缺陷病毒（HIV），随后病毒发生逃逸，但用两条 gRNA 的特定组合可完全使具有感染性的 HIV 失活。新型 RNA 导向内切酶系统 CRISPR-Cas12a（以前称为 Cpf1）可能为基因组工程提供更有前途的工具，其活性和特异性更高。我们比较了 Cas12a 与原始 Cas9 系统对整合 HIV DNA 基因组的失活作用。据报道，Cas12a 的抗病毒活性更高，仅用一条 gRNA（称为 crRNA）即可实现完全的 HIV 失活。我们提出，Cas9 与 Cas12a 内切酶的不同结构解释了这种效应。我们还披露，Cas12a 内切酶的 DNA 切割和随后的 DNA 修复会导致具有与 Cas9 不同序列特征的突变。两种 CRISPR 系统都可以诱导 DNA 切割和随后修复部位的典型小缺失，但 Cas12a 不会诱导 Cas9 常规观察到的纯 DNA 插入。尽管这些典型特征在许多文献研究中很明显，但这是首次记录这些显著差异的报告。
