Targeted mutagenesis of the SUMO protease, Overly Tolerant to Salt1 in rice through CRISPR/Cas9-mediated genome editing reveals a major role of this SUMO protease in salt tolerance.

Abstract

SUMO proteases are encoded by a large gene family in rice and are a potential source of specificity within the SUMO system that is responding to different environmental cues. We previously demonstrated a critical role of OsOTS class of SUMO proteases in salt and drought stress in rice by silencing several family members collectively via RNAi methods. However, to date it has not been possible to assign a role to specific family members due to limitations of RNAi mediated off target silencing across several members of the gene family. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease 9 (CRISPR/Cas9) system has emerged as a promising technology for specific gene editing in crop plants. Here, we demonstrate targeted mutagenesis of OsOTS1 in rice using the CRISPR/Cas9 gene editing system in the rice cultivar Kitaake. Guide RNA mediated mutations in OsOTS1 was highly efficient with almost 95% of T0 transgenics showing the desired effect with no off-target mutations. The OsOTS1 mutations observed in T0 generation were heritable in subsequent generations. OsOTS1 CRISPR lines show enhanced sensitivity to salt with reduced root and shoot biomass indicating that OsOTS1 has a major role in salt stress tolerance in rice. This unexpected finding indicates that precise and effective genome editing can be used to characterise specificity within the SUMO system in rice.