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

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 expre...

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Autores principales: Butt, Haroon, Piatek, Agnieszka, Li, Lixin, S. N. Reddy, Anireddy, M. Mahfouz, Magdy
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6723545/
https://www.ncbi.nlm.nih.gov/pubmed/31394891
http://dx.doi.org/10.3390/genes10080596
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author Butt, Haroon
Piatek, Agnieszka
Li, Lixin
S. N. Reddy, Anireddy
M. Mahfouz, Magdy
author_facet Butt, Haroon
Piatek, Agnieszka
Li, Lixin
S. N. Reddy, Anireddy
M. Mahfouz, Magdy
author_sort Butt, Haroon
collection PubMed
description 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.
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spelling pubmed-67235452019-09-10 Multiplex CRISPR Mutagenesis of the Serine/Arginine-Rich (SR) Gene Family in Rice Butt, Haroon Piatek, Agnieszka Li, Lixin S. N. Reddy, Anireddy M. Mahfouz, Magdy Genes (Basel) Article 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. MDPI 2019-08-07 /pmc/articles/PMC6723545/ /pubmed/31394891 http://dx.doi.org/10.3390/genes10080596 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Butt, Haroon
Piatek, Agnieszka
Li, Lixin
S. N. Reddy, Anireddy
M. Mahfouz, Magdy
Multiplex CRISPR Mutagenesis of the Serine/Arginine-Rich (SR) Gene Family in Rice
title Multiplex CRISPR Mutagenesis of the Serine/Arginine-Rich (SR) Gene Family in Rice
title_full Multiplex CRISPR Mutagenesis of the Serine/Arginine-Rich (SR) Gene Family in Rice
title_fullStr Multiplex CRISPR Mutagenesis of the Serine/Arginine-Rich (SR) Gene Family in Rice
title_full_unstemmed Multiplex CRISPR Mutagenesis of the Serine/Arginine-Rich (SR) Gene Family in Rice
title_short Multiplex CRISPR Mutagenesis of the Serine/Arginine-Rich (SR) Gene Family in Rice
title_sort multiplex crispr mutagenesis of the serine/arginine-rich (sr) gene family in rice
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6723545/
https://www.ncbi.nlm.nih.gov/pubmed/31394891
http://dx.doi.org/10.3390/genes10080596
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