Multiplexed conditional genome editing with Cas12a in Drosophila

Area of Science:

• Genetics
• Molecular Biology
• Biotechnology

Background:

• CRISPR-Cas9 is the primary gene editing tool in Drosophila melanogaster.
• Expanding the CRISPR toolkit with new nucleases can enhance genome engineering capabilities.
• Cas12a offers potential advantages over Cas9 for targeted genome modification.

Purpose of the Study:

• To establish and characterize a CRISPR-Cas12a gene editing platform in Drosophila.
• To evaluate the efficiency and regulation of LbCas12a in vivo.
• To demonstrate the utility of Cas12a for multiplex and conditional genome engineering.

Main Methods:

• Utilized Cas12a from Lachnospiraceae bacterium for gene editing in Drosophila.
• Investigated temperature-dependent activity of LbCas12a.
• Employed CRISPR RNA (crRNA) arrays for multiplex editing.
• Implemented conditional expression systems for tissue-specific gene editing.
• Tested a D156R mutant LbCas12a variant for enhanced activity.

Main Results:

• LbCas12a mediates robust in vivo gene editing in Drosophila.
• Cas12a activity is modulated by temperature (high at 29°C, low at 18°C).
• LbCas12a efficiently utilizes compact crRNA arrays for multiplex genome engineering.
• Conditional LbCas12a expression enables tightly controlled, tissue-specific gene editing.
• A D156R LbCas12a variant exhibits higher activity and outperforms Cas9 in essential gene identification.

Conclusions:

• Cas12a gene editing expands the genome engineering toolbox in Drosophila.
• This system is a powerful method for functional genome annotation.
• A genetically encoded Cas12a system in an animal is presented, enabling multiplexed, conditional genome engineering.