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Vol 59(2025) N 2 p. 224-233; DOI 10.1134/S0026893324700857 ![]() J.A. Taran1*, R.R. Mintaev1, D.V. Glazkova1, B.V. Belugin1, E.V. Bogoslovskaya1, G.A. Shipulin1 Influence of Homology Arm Length and Structure on the Efficiency of Long Transgene Integration into a Cleavage Site Induced by SpCas9 or AsCpf1 1Center for Strategic Planning, Federal Medical and Biological Agency, Moscow, 119121 Russia*taran.julia01@gmail.com Received - 2024-08-01; Revised - 2024-10-07; Accepted - 2024-10-07 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%. HIV, CRISPR/Cas, CCR5, homologous recombination |