Taran J A, Mintaev R R, Glazkova D V, Belugin B V, Bogoslovskaya E V, Shipulin G A
Center for Strategic Planning, Federal Medical and Biological Agency, Moscow, 119121 Russia.
Mol Biol (Mosk). 2025 Mar-Apr;59(2):255-265.
One of the promising new approaches to the treatment of HIV infection is CRISPR/Cas-mediated knockout of the CCR5 receptor gene followed by the integration of an anti-HIV gene into the break site. Numerous studies have focused on the knockout of the CCR5 gene; however, the efficiency of subsequent targeted integration of long fragments remains poorly studied. To evaluate the efficiency of this approach, we used HT1080 cells and investigated the integration of a cassette expressing the EGFP gene into the CCR5 locus using two different nucleases (SpCas9 and AsCpf1) and various donor DNA constructs delivered by recombinant adeno-associated viral vectors (rAAV). For each nuclease, we designed five variants of donor DNA differing in the length (ranging from 150 to 1000 bp) or structure of the homology arms. The efficiency of transgene integration with 150 bp homology arms was the lowest for both nucleases and differed significantly from constructs with longer homology arms. Furthermore, it was shown that the presence of nuclease cleavage sites in the donor DNA flanking the cassette with homology arms did not affect the efficiency of transgene integration during AAV delivery. We demonstrated that the AsCpf1 nuclease provided higher efficiency of EGFP transgene integration than SpCas9, despite the lower efficiency of CCR5 knockout. The maximum percentage of cells with the integrated transgene was achieved using the AsCpf1 nuclease and an expression cassette with 600 bp homology arms, reaching 59 ± 6%.
治疗HIV感染的一种有前景的新方法是通过CRISPR/Cas介导敲除CCR5受体基因，然后将抗HIV基因整合到断裂位点。众多研究聚焦于CCR5基因的敲除；然而，后续长片段靶向整合的效率仍研究不足。为评估该方法的效率，我们使用HT1080细胞，利用两种不同的核酸酶（SpCas9和AsCpf1）以及由重组腺相关病毒载体（rAAV）递送的各种供体DNA构建体，研究了表达EGFP基因的盒式结构整合到CCR5基因座的情况。对于每种核酸酶，我们设计了五种供体DNA变体，其同源臂的长度（范围从150到1000 bp）或结构不同。对于两种核酸酶而言，具有150 bp同源臂的转基因整合效率最低，且与具有更长同源臂的构建体有显著差异。此外，研究表明，在带有同源臂的盒式结构侧翼的供体DNA中存在核酸酶切割位点，在AAV递送过程中并不影响转基因整合效率。我们证明，尽管CCR5敲除效率较低，但AsCpf1核酸酶提供的EGFP转基因整合效率高于SpCas9。使用AsCpf1核酸酶和具有600 bp同源臂的表达盒式结构，实现了整合转基因的细胞的最大百分比，达到59±6%。
