An intein-mediated split-nCas9 system for base editing in plants

Abstract

Virus-assisted delivery of the clustered regularly interspaced short palindromic (CRISPR)/CRISPR-associated (Cas) system represents a promising approach for editing plant genomes. However, the relatively large size of the CRISPR/Cas9 system is challenging to package into viral vectors with confined packaging capacity. To address this technical challenge, we developed a strategy that splits the required CRISPR-Cas9 components across a dual-vector system in which CRISPR-Cas reassembles into an active form following co-infection to achieve targeted genome editing in plant cells. An intein-mediated split system was adapted and optimized in plant cells by successfully demonstrating split-eYGFPuv expression. Using a plant-based biosensor, we demonstrated for the first time that the split-SpnCas9 is capable of inducing efficient base editing in plant cells and identified several valid split sites for future biodesign strategies. Overall, this strategy provides new opportunities to bridge different CRISPR/Cas9 tools including base editor, prime editor, and CRISPR activation with virus-mediated gene editing.