Enhanced cleavage of genomic <i>CCR5</i> using CasX2 ^Max

Development of novel CRISPR/Cas systems enhances opportunities for gene editing to treat infectious diseases, cancer, and genetic disorders. CasX2 (<i>Plm</i>Cas12e) belongs to the class II CRISPR system derived from <i>Planctomycetes</i>, a non-pathogenic bacterium present in aquatic and terrestrial soils and offers several advantages as a potential therapeutic CRISPR system over <i>Streptococcus pyogenes</i> Cas9 (<i>Sp</i>Cas9) and <i>Staphylococcus aureus</i> Cas9 (<i>Sa</i>Cas9). These advantages include its smaller size, distinct protospacer adjacent motif (PAM) requirements, staggered cleavage cuts that promote homology-directed repair, and the absence of pre-existing immunity in humans. We compared the cleavage efficiency and double-stranded break repair characteristics between CasX2 and CasX2^Max, a recently generated CasX2 variant with three amino acid substitutions, for targeting <i>CCR5</i>, a gene that encodes the CCR5 receptor important for HIV-1 infection. Two single guide RNAs (sgRNAs) were designed that flank the 32 bases deleted in the natural <i>CCR5 ∆32</i> mutation. Nanopore sequencing demonstrated that CasX2 using sgRNAs with spacers of 17 nucleotides (nt), 20 nt or 23 nt in length were ineffective at cleaving genomic <i>CCR5</i>. In contrast, CasX2^Max using sgRNAs with 20 nt and 23 nt spacer lengths, enabled cleavage of genomic <i>CCR5</i>. Structural modelling indicated that two of the CasX2^Max amino acid substitutions enhanced sgRNA-DNA duplex stability, while the third improved DNA strand alignment within the catalytic site. These structural changes likely underlie the increased activity of CasX2^Max in cellular gene excision. In sum, CasX2^Max consistently outperformed native CasX2 across all assays and represents a superior gene-editing platform for therapeutic applications.