Engineered Cas12a-Plus nuclease enables gene editing with enhanced activity and specificity

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• CRISPR-Cas12a is a gene-editing tool with unique features like T-rich PAM recognition and sticky-end DNA cleavage.
• Distinct from Cas9, Cas12a offers broader targeting and potential for accurate genome editing.
• Current Cas12a variants often compromise specificity for higher activity, limiting clinical applications.

Purpose of the Study:

• To engineer enhanced CRISPR-Cas12a variants with improved activity and specificity.
• To develop novel AsCas12a and LbCas12a variants for more precise genome editing.
• To create tools suitable for clinical applications by balancing editing efficiency and accuracy.

Main Methods:

• Structure-guided protein engineering was employed to modify Acidaminococcus sp. Cas12a (AsCas12a) and Lachnospiraceae bacterium Cas12a (LbCas12a).
• New variants, AsCas12a-Plus and LbCas12a-Plus, were created and characterized for editing efficiency and specificity.
• Performance was evaluated in multiplex editing, transcriptional activation, and disruption of the BRAF^V600E oncogene.

Main Results:

• AsCas12a-Plus demonstrated 1.5–2.0-fold increased activity and reduced off-target mutations (from 29 to 23).
• LbCas12a-Plus showed ~1.1-fold activity improvement and decreased off-targets (from 20 to 12), with enhanced specificity.
• AsCas12a-Plus maintained efficiency and reduced off-target activity when disrupting BRAF^V600E, outperforming other variants.

Conclusions:

• Developed AsCas12a-Plus and LbCas12a-Plus variants offer superior specificity and activity for gene editing.
• These engineered Cas12a variants expand the gene-editing toolbox.
• The enhanced variants show promise for increased suitability in clinical applications.