Discovery and engineering of ChCas12b for precise genome editing

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR-Cas12b nucleases offer potential for genome editing but require further exploration.
• Computational identification of numerous CRISPR-Cas12b variants has outpaced functional characterization.

Purpose of the Study:

• To screen CRISPR-Cas12b nucleases for mammalian genome editing applications.
• To characterize a novel Cas12b variant with an expanded targeting scope and high fidelity.

Main Methods:

• A GFP-activation assay was employed to screen 13 Cas12b nucleases.
• Functional characterization of promising candidates, including ChCas12b, across nine endogenous loci.
• Identification and validation of mutations enhancing ChCas12b specificity and enabling allele-specific gene disruption.

Main Results:

• Five active Cas12b candidates were identified from the screen.
• Candidatus hydrogenedentes Cas12b (ChCas12b) demonstrated a WTN PAM, significantly broadening targeting possibilities.
• Optimized ChCas12b showed SpCas9-comparable activity and enabled allele-specific disruption of SNPs, even with a high-fidelity variant.

Conclusions:

• ChCas12b is a versatile CRISPR-Cas12b tool with an expanded PAM recognition and high editing efficiency.
• Engineered ChCas12b variants offer enhanced specificity and allele-specific editing capabilities.
• ChCas12b and its variants represent valuable tools for fundamental research and potential therapeutic genome editing applications.