Engineering of CRISPR-Cas12b for human genome editing

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Type-V CRISPR-Cas12b effectors are challenging for human genome editing due to high temperature requirements.
• Existing Cas12b variants exhibit limitations in human cell editing efficiency.

Purpose of the Study:

• To explore Cas12b family diversity and identify a suitable candidate for human gene editing.
• To engineer gain-of-function mutations in Bacillus hisashii Cas12b (BhCas12b) to enhance its genome editing capabilities in human cells.

Main Methods:

• Bioinformatic exploration of Cas12b family diversity.
• Characterization of wild-type and mutant BhCas12b activity at 37°C in human cell lines.
• Assessment of genome editing efficiency and specificity in human cell lines and primary T cells.

Main Results:

• Identified BhCas12b from Bacillus hisashii as a promising Cas12b candidate.
• Wild-type BhCas12b showed preferential nicking, limiting double-strand break formation at 37°C.
• Gain-of-function mutations enabled robust and specific genome editing with BhCas12b in human cells and primary T cells, outperforming S. pyogenes Cas9 in specificity.

Conclusions:

• Engineered BhCas12b represents a novel and efficient RNA-guided nuclease platform for human genome editing.
• This provides a third major CRISPR system, alongside Cas9 and Cas12a, for therapeutic and research applications.
• Mutant BhCas12b offers enhanced specificity compared to S. pyogenes Cas9 for precise genome engineering.