A Potent CRISPR-Cas12l Double-Strand Break Gene Editor.

Area of Science:

• CRISPR-Cas systems biology
• Molecular biology and genetics
• Protein engineering and design

Background:

• Discovery of a novel CRISPR-Cas12l family from the Armatimonadota phylum.
• Cas12l nucleases are compact, recognize a 5' C-rich PAM, and possess a unique N-terminal DNA-binding domain.
• Existing CRISPR tools require optimization for enhanced genome editing efficiency and specificity.

Purpose of the Study:

• To engineer Asp2Cas12l variants with improved DNA target cleavage rates.
• To enhance double-strand break (DSB) editing efficiency and reduce target-to-target variation.
• To evaluate the performance of engineered Cas12l in human cells for genome editing.

Main Methods:

• Structure-guided rational protein design.
• AI-based large protein language model predictions for variant design.
• Assessment of DSB editing efficiency in human cells.
• Analysis of homology-directed repair (HDR) outcomes.

Main Results:

• Engineered Asp2Cas12l variants demonstrated approximately a 10-fold increase in DSB editing efficiency compared to wild-type.
• Reduced target-to-target variation observed in engineered variants.
• Editing frequencies were comparable to SpCas9 at overlapping target sites.
• Efficient correction of DSBs by homology-directed repair (39-56% of editing outcomes).

Conclusions:

• This study successfully engineered Cas12l variants with significantly enhanced genome editing capabilities.
• The engineered Cas12l nucleases represent a potent new alternative for DSB-mediated genome editing in human cells.
• Findings advance the understanding of CRISPR-Cas12 protein engineering and its applications.