Experimental Selection of Effective sgRNAs for MmCas12m Targeting the Region of the Start Codon of the HIV-1 gag Gene

HIV-1 remains a threat to global health. There is no effective vaccine or drug for a complete cure of HIV infection. Work continues on the development of gene therapy drugs against HIV-1. The use of DNA base editors delivered to the editing site by CRISPR-Cas systems has shown some success in the field of HIV-1 gene therapy. The MmCas12m isoform obtained from Mycolicibacterium mucogenicum can become a promising platform for this task. MmCas12m has a compact size, the ability to bind strongly to the target DNA sequence, and an absence of nuclease activity. Thus, MmCas12m can act not only as a platform for the delivery of DNA base editors, but also as an inhibitor of transcription from HIV-1 proviral DNA. We experimentally selected in vitro the most effective sgRNAs for MmCas12m to target the start codon of the gag HIV-1, the product of which is important in virion assembly. Of the nine sgRNAs we selected, four showed statistically significant effectiveness in targeting the desired region of the HIV-1 genome. To test the effectiveness of each sgRNA, we have developed a system suitable for evaluating the binding of any Cas protein to the target site of HIV-1 genome editing.