Establishing Programmable CRISPR/Cas13b-Mediated Knockdown System in Rice

Abstract

CRISPR-Cas endonucleases mediate prokaryotic adaptive immunity by targeting foreign nucleic acids. CRISPR/Cas13b is a class 2 type VI-B ribonuclease that targets and cleaves single-stranded RNA. It exhibits higher RNA interference activity than Cas13a and Cas13c and causes fewer collateral effects than RxCas13d in mammalian cells. However, a programmable CRISPR/Cas13b-mediated RNA interference system for endogenous transcripts in rice has not yet been established. Here, we developed a CRISPR/Cas13b-mediated system to target endogenous transcripts in rice. Our CRISPR/Cas13b system could inhibit multiple endogenous mRNAs simultaneously. In addition, this system efficiently repressed endogenous long noncoding RNAs with more than 50% inhibition in stable transgenic plants. Furthermore, we found only weak collateral effects of the CRISPR/Cas13b-mediated system at the transcriptome-wide level, and no difference in the agronomic traits of stable transgenic rice in the field. We present a programmable CRISPR/Cas13b-mediated knockdown system for rice, offering a potential biotechnological tool for functional genomics and crop improvement.