Enhanced cleavage of genomic CCR5 using CASX2^Max

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biochemistry

Background:

• CRISPR/Cas systems offer powerful gene editing capabilities for various diseases.
• CasX2 (PlmCas12e) presents advantages over SpCas9 and SaCas9 for therapeutic use due to its size, PAM specificity, cleavage pattern, and lack of pre-existing immunity.
• The CCR5 receptor is a key target for HIV-1 infection, making its gene a relevant target for gene editing therapies.

Purpose of the Study:

• To compare the gene-editing efficiency of CasX2 and its variant CasX2Max.
• To investigate the double-stranded break repair characteristics of CasX2 and CasX2Max.
• To evaluate the potential of CasX2Max as a therapeutic gene-editing platform targeting the CCR5 gene.

Main Methods:

• Utilized CRISPR/Cas gene editing systems, CasX2 and CasX2Max, with engineered single guide RNAs (sgRNAs).
• Targeted the CCR5 gene, specifically flanking the region of the natural CCR5 ∆32 mutation.
• Employed Nanopore sequencing to assess cleavage efficiency and analyzed structural modeling to understand variant function.

Main Results:

• Native CasX2 was ineffective in cleaving genomic CCR5 across tested sgRNA spacer lengths (17-23 nt).
• CasX2Max demonstrated successful cleavage of genomic CCR5 using sgRNAs with 20 nt and 23 nt spacer lengths.
• Structural modeling revealed that amino acid substitutions in CasX2Max enhance sgRNA-DNA stability and DNA alignment in the catalytic site, leading to improved activity.

Conclusions:

• CasX2Max significantly outperforms native CasX2 in gene editing assays, particularly for targeting the CCR5 gene.
• The structural modifications in CasX2Max are responsible for its enhanced cleavage efficiency.
• CasX2Max represents a promising and superior gene-editing platform for therapeutic applications, including the potential treatment of HIV-1 infection.