<br>CRISPR-Cas12b 能够高效攻击 T 细胞培养物中的 HIV 前病毒 DNA,Biomedicine & Pharmacotherapy

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CRISPR-Cas12b 能够高效攻击 T 细胞培养物中的 HIV 前病毒 DNA
Biomedicine & Pharmacotherapy ( IF 6.9 ) Pub Date : 2023-06-28 , DOI: 10.1016/j.biopha.2023.115046
Minghui Fan ₁ , Yuanling Bao ₁ , Ben Berkhout ₁ , Elena Herrera-Carrillo ₁

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新型核酸内切酶 Cas12b 专为哺乳动物细胞中的靶向基因组编辑而设计，由于其尺寸小、序列特异性高且能够产生相对较大的缺失，因此在某些应用中是一种有前途的工具。我们之前报道过 spCas9 和 Cas12a 攻击整合的病毒 DNA 基因组后，细胞培养物感染中的人类免疫缺陷病毒 (HIV) 受到抑制。现在，我们使用抗 HIV gRNA 测试了 Cas12b 核酸内切酶抑制细胞培养物中 HIV 感染传播的能力。在长期的 HIV 复制研究中测试了病毒抑制，这使我们能够测试病毒逃逸以及治愈受感染 T 细胞的可能性。我们证明 Cas12b 仅用一个 gRNA 即可实现 HIV 完全灭活，而 Cas9 需要两个 gRNA 才能实现这一结果。当Cas12b系统被编程为两种抗病毒gRNA时，整体抗HIV效力得到提高，并且由于多重切割修复作用而产生更严重突变的HIV原病毒。由于HIV基因组的多个重要部分发生突变，这种“超突变”的HIV原病毒更有可能存在缺陷。我们报告Cas9、Cas12a和Cas12b核酸内切酶的突变谱存在显着差异，这可能对病毒灭活水平产生影响。这些综合结果使 Cas12b 成为 HIV 灭活的首选编辑系统。这些结果为 CRISPR-Cas12b 介导的 HIV-1 灭活提供了体外“概念证明”。

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CRISPR-Cas12b enables a highly efficient attack on HIV proviral DNA in T cell cultures

The novel endonuclease Cas12b was engineered for targeted genome editing in mammalian cells and is a promising tool for certain applications because of its small size, high sequence specificity and ability to generate relatively large deletions. We previously reported inhibition of the human immunodeficiency virus (HIV) in cell culture infections upon attack of the integrated viral DNA genome by spCas9 and Cas12a. We now tested the ability of the Cas12b endonuclease to suppress a spreading HIV infection in cell culture with anti-HIV gRNAs. Virus inhibition was tested in long-term HIV replication studies, which allowed us to test for viral escape and the potential for reaching a CURE of the infected T cells. We demonstrate that Cas12b can achieve complete HIV inactivation with only a single gRNA, a result for which Cas9 required two gRNAs. When the Cas12b system is programmed with two antiviral gRNAs, the overall anti-HIV potency is improved and more grossly mutated HIV proviruses are generated as a result of multiple cut-repair actions. Such “hypermutated” HIV proviruses are more likely to be defective due to mutation of multiple essential parts of the HIV genome. We report that the mutational profiles of the Cas9, Cas12a and Cas12b endonucleases differ significantly, which may have an impact on the level of virus inactivation. These combined results make Cas12b the preferred editing system for HIV-inactivation. These results provide in vitro “proof of concept’ for CRISPR-Cas12b mediated HIV-1 inactivation.

更新日期：2023-06-28

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