Intein-mediated split SaCas9 for genome editing in plants

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Genetics

Background:

• Plant genome editing faces limitations due to reliance on genetic transformation and regeneration.
• Large gene editing nucleases like Cas9 and Cas12a pose challenges for Virus-Induced Genome Editing (VIGE) applications in plants.

Purpose of the Study:

• To overcome the size limitations of nucleases in VIGE by developing an intein-mediated split SaCas9 system.
• To assess the efficiency of the split SaCas9 system in both transgenic plants and challenging species like sheepgrass using VIGE.

Main Methods:

• Intein-mediated protein splicing was used to create split SaCas9 systems (Split-v1 and Split-v3).
• Barley stripe mosaic virus (BSMV)-based vectors were constructed for co-expression of Split-v1 SaCas9 and guide RNAs (gRNAs).
• VIGE was applied to sheepgrass (Leymus chinensis) targeting LcHRC, LcGW2, and LcTB1 genes.

Main Results:

• The Split-v1 system achieved high genome editing efficiencies (70.2%–96.1%) in transgenic plants, comparable to wild-type SaCas9.
• VIGE using Split-v1 SaCas9 in sheepgrass resulted in genome editing efficiencies ranging from 10.40% to 37.03%.
• Successful application of VIGE in recalcitrant sheepgrass demonstrates the system's potential.

Conclusions:

• Intein-mediated split nucleases offer a viable strategy to overcome nuclease size constraints in VIGE.
• This approach significantly broadens the applicability of VIGE, particularly in plant species that are difficult to transform.
• The developed Split-v1 system shows promise for efficient genome editing in diverse plant systems.