Enhanced cleavage of genomic CCR5 using CasX2Max
Christine A. Hodge, Niles P. Donegan, David A. Armstrong, Matthew S. Hayden, Alexandra L. Howell
TL;DR
Researchers improved the CasX2 CRISPR system to more effectively edit the CCR5 gene, which is important for HIV-1 infection.
Contribution
A new CasX2 variant, CasX2Max, was developed with enhanced genomic CCR5 cleavage efficiency.
Findings
CasX2Max enabled cleavage of genomic CCR5 with 20 nt and 23 nt sgRNAs, unlike native CasX2.
Structural modeling showed that CasX2Max substitutions improved sgRNA-DNA stability and DNA alignment.
CasX2Max outperformed native CasX2 in all tested gene-editing assays.
Abstract
Development of novel CRISPR/Cas systems enhances opportunities for gene editing to treat infectious diseases, cancer, and genetic disorders. CasX2 (PlmCas12e) belongs to the class II CRISPR system derived from Planctomycetes, a non-pathogenic bacterium present in aquatic and terrestrial soils and offers several advantages as a potential therapeutic CRISPR system over Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9). 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 CasX2Max, a recently generated CasX2 variant with three amino acid substitutions, for targeting CCR5, a gene that encodes the CCR5…
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Taxonomy
TopicsCRISPR and Genetic Engineering · RNA and protein synthesis mechanisms · Animal Genetics and Reproduction