Cas12a variants designed for lower genome-wide off-target effect through stringent PAM recognition

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• Cas12a is an RNA-guided endonuclease utilized for multiplex gene editing.
• Minimizing off-target effects is crucial for safe and effective gene editing.
• Existing Cas12a variants offer convenience but can still exhibit off-target activity.

Purpose of the Study:

• To engineer Cas12a variants with improved specificity and reduced off-target effects.
• To investigate the impact of modified PAM recognition on Cas12a activity and specificity.
• To assess the gene-editing capabilities of novel Cas12a variants in mammalian cells.

Main Methods:

• Engineering of LbCas12a (Lb-K538R) and Lb2Cas12a (Lb2-K518R) variants with altered Protospacer Adjacent Motif (PAM) recognition.
• Evaluation of gene-editing efficiency and off-target effects in mammalian cells.
• Genome-wide off-target analysis of engineered Cas12a variants, including As-K548R.

Main Results:

• Engineered Lb-K538R and Lb2-K518R variants demonstrated more stringent PAM recognition (YYN to TYN) and reduced off-target effects compared to wild-type Cas12a.
• Lb2Cas12a exhibited extensive gene-editing activities in mammalian cells.
• The As-K548R variant also showed decreased off-target effects in genome-wide analysis.
• Modified Cas12a variants maintained similar editing efficiency in vivo.

Conclusions:

• Cas12a proteins can be rationally designed for stringent PAM recognition to minimize off-target effects.
• Engineered Cas12a variants provide a more precise and potentially safer tool for genome editing.
• This approach offers a practical strategy for enhancing the specificity of gene editing technologies.