Hs1Cas12a and Ev1Cas12a confer efficient genome editing in plants

Area of Science:

• Molecular Biology
• Biotechnology
• Plant Science

Background:

• CRISPR-Cas12a (Cpf1) is a versatile genome editing tool, particularly effective in AT-rich regions.
• Expanding the CRISPR-Cas12a toolbox with novel orthologs is crucial for diverse plant applications.

Purpose of the Study:

• To discover and characterize novel Cas12a orthologs for plant genome editing.
• To evaluate the efficiency and characteristics of new Cas12a variants in monocot and dicot species.

Main Methods:

• Screening of 17 novel Cas12a orthologs for genome editing activity.
• Testing selected orthologs (Ev1Cas12a, Hs1Cas12a) in rice and tomato protoplasts.
• Assessing editing efficiency, biallelic editing, and chimerism in T0 plants (rice, poplar).

Main Results:

• Ev1Cas12a and Hs1Cas12a demonstrated efficient multiplexed genome editing in rice and tomato protoplasts.
• Hs1Cas12a showed enhanced performance at lower temperatures and achieved 87.5% biallelic editing in rice.
• Both enzymes effectively edited poplar T0 plants, reaching 100% edited plants, though with high chimerism.

Conclusions:

• Ev1Cas12a and Hs1Cas12a are potent genome editing tools for both monocot (rice) and dicot (tomato, poplar) plants.
• These novel Cas12a orthologs significantly enrich the available toolkit for plant genetic engineering.
• The findings suggest broad applicability of Ev1Cas12a and Hs1Cas12a in diverse plant species and future biotechnological advancements.