The low efficiency of knock-in, especially in primary human cells, limits the use of genome editing technology for therapeutic purposes, which makes it important to develop approaches for increasing knock-in levels. In this work, using a knock-in model of the peptide fusion inhibitor of HIV MT-C34 into the human CXCR4 locus in the CEM/R5 T cell line, we analyzed the effectiveness of several approaches to increasing knock-in levels. First, donor DNA modification aimed at improving the efficiency of plasmid transport into the nucleus was evaluated, namely the introduction into the donor plasmid of the SV40 DNA transport sequence (DTS) or the binding sites for the transcription factor NF-κB, whose effects on knock-in levels have not been described. In the MT-C34 knock-in model into the CXCR4 locus, this modification was ineffective. The second approach, modifying the Cas9 nuclease by introducing two additional nuclear localization signals (NLS), increased the knock-in level by 30%. Finally, blocking DNA repair via the nonhomologous end joining pathway using DNA-dependent protein kinase inhibitors caused a 1.8-fold increase in knock-in. The combination of the last two approaches caused an additive effect. Thus, increasing the number of NLSs in the Cas9 protein and inhibiting DNA repair via the nonhomologous end joining pathway significantly increased the level of knock-in of the HIV-1 peptide fusion inhibitor into the clinically relevant locus CXCR4, which can be used to develop effective gene therapy approaches for the treatment of HIV infection.
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