ENHANCED CLEAVAGE OF GENOMIC CCR5 USING CASX2^Max

Development of novel CRISPR/Cas systems enhances opportunities for gene editing to treat infectious diseases, cancer, and genetic disorders. We evaluated CasX2 (PlmCas12e), a class II CRISPR system derived from Planctomycetes, a non-pathogenic bacterium present in aquatic and terrestrial soils. CasX2 offers several advantages over Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9), including its smaller size, distinct protospacer adjacent motif (PAM) requirements, staggered cleavage cuts that promote homology-directed repair, and no known pre-existing immunity in humans. A recent study reported that a three amino acid substitution in CasX2 significantly enhanced cleavage activity (1). Therefore, we compared cleavage efficiency and double-stranded break repair characteristics between the native CasX2 and the variant, CasX2^Max, for cleavage of CCR5, a gene that encodes the CCR5 receptor important for HIV-1 infection. Two CasX2 single guide RNAs (sgRNAs) were designed that flanked the 32 bases deleted in the natural CCR5 Δ32 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 CCR5. In contrast, CasX2^Max using sgRNAs with 20 nt and 23 nt spacer lengths, enabled robust genomic cleavage of CCR5. Structural modeling indicated that two of the CasX2^Max 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.