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Open AccessArticle

Multiplex CRISPR Mutagenesis of the Serine/Arginine-Rich (SR) Gene Family in Rice

1
Laboratory for Genome Engineering and Synthetic Biology, Division of Biological Sciences, King Abdullah University of Science and Technology, Thuwal 23955-6900, Saudi Arabia
2
Program in Molecular Plant Biology, Program in Cell and Molecular Biology, Department of Biology, Colorado State University, Fort Collins, CO 80523-1062, USA
*
Author to whom correspondence should be addressed.
Genes 2019, 10(8), 596; https://doi.org/10.3390/genes10080596
Received: 25 June 2019 / Revised: 19 July 2019 / Accepted: 5 August 2019 / Published: 7 August 2019
(This article belongs to the Special Issue TILLING and CRISPR to design the varieties of tomorrow)
Plant growth responds to various environmental and developmental cues via signaling cascades that influence gene expression at the level of transcription and pre-mRNA splicing. Alternative splicing of pre-mRNA increases the coding potential of the genome from multiexon genes and regulates gene expression through multiple mechanisms. Serine/arginine-rich (SR) proteins, a conserved family of splicing factors, are the key players of alternative splicing and regulate pre-mRNA splicing under stress conditions. The rice (Oryza sativa) genome encodes 22 SR proteins categorized into six subfamilies. Three of the subfamilies are plant-specific with no mammalian orthologues, and the functions of these SR proteins are not well known. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a genome engineering tool that cleaves the target DNA at specific locations directed by a guide RNA (gRNA). Recent advances in CRISPR/Cas9-mediated plant genome engineering make it possible to generate single and multiple functional knockout mutants in diverse plant species. In this study, we targeted each rice SR locus and produced single knockouts. To overcome the functional redundancy within each subfamily of SR genes, we utilized a polycistronic tRNA-gRNA multiplex targeting system and targeted all loci of each subfamily. Sanger sequencing results indicated that most of the targeted loci had knockout mutations. This study provides useful resource materials for understanding the molecular role of SR proteins in plant development and biotic and abiotic stress responses. View Full-Text
Keywords: splicing; alternative splicing; SR proteins; genome engineering; multiplex targeting; CRISPR/Cas9 splicing; alternative splicing; SR proteins; genome engineering; multiplex targeting; CRISPR/Cas9
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Butt, H.; Piatek, A.; Li, L.; S. N. Reddy, A.; M. Mahfouz, M. Multiplex CRISPR Mutagenesis of the Serine/Arginine-Rich (SR) Gene Family in Rice. Genes 2019, 10, 596.

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