Investigating The Molecular Functions of The Os-Sc106 Spliceosomal Protein Via CRISPR/Cas9 System

Abstract

Plants employ sophisticated molecular machineries to fine-tune their responses to growth, developmental, and stress cues. Plants cellular response influences gene expression through regulating processes like transcription and splicing. To increase the genome coding potential and further regulate the expression, pre-mRNA is alternatively spliced. Serine/Arginine-rich (SR) proteins, a family of pre-mRNA splicing factors, recognize splicing cis-elements and regulate both constitutive and alternative splicing. Recent studies reported only 22 SR proteins encoded in the genome of rice (Oryza sativa), which are classified into 6 subfamilies. Oryza s. SC subfamily 106 kDa (Os-Sc106) locus is homologous to the human SR protein SFSR11 (SRp54). Os-Sc106 contains SR proteins characteristics, and was not included among the rice SR proteins. The clustered regularly interspaced short palindromic repeats (CRISPR) and its associated protein 9 (Cas9) system, an RNA-guided endonuclease complex that introduces a double-strand break (DSB) into the DNA. Innovative scientific advances in genome engineering have made CRISPR/Cas9 an excellent system to conduct functional knockout studies of genes in most biological systems including plants. In this study, I targeted the rice Os-Sc106 locus at exon1, and 3 via CRISPR/Cas9 system. Genotyping analyses revealed the recovery of Os-Sc106 mutants including complete functional knockouts such as sf11h-2, sf11h-8, and sf11h-55. Phenotypic analyses show that Os-Sc106 mutants (sf11h-2, 8, 55, and 57) are oversensitive under abiotic stress in comparison to WT plants, suggesting that Os-Sc106 locus encodes a protein that is important for regulating plant stress responses.