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

Genes ◽  
2019 ◽  
Vol 10 (8) ◽  
pp. 596 ◽  
Author(s):  
Haroon Butt ◽  
Agnieszka Piatek ◽  
Lixin Li ◽  
Anireddy S. N. Reddy ◽  
Magdy M. Mahfouz
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Stress Responses ◽  
Genome Engineering ◽  
Sr Proteins ◽  
Mrna Splicing ◽  
Plant Genome ◽  
Biotic And Abiotic Stress ◽  
Guide Rna ◽  
A Genome

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.

scholarly journals A multiplex guide RNA expression system and its efficacy for plant genome engineering

2020 ◽  
Author(s):  
Youngbin Oh ◽  
Hyeonjin Kim ◽  
Bora Lee ◽  
Sang-Gyu Kim
Keyword(s):  
Genome Engineering ◽  
Binary Vector ◽  
Expression System ◽  
Plant Genome ◽  
Nicotiana Attenuata ◽  
Specific Sequence ◽  
Editing Efficiency ◽  
Guide Rna ◽  
Assembly Method ◽  
A Genome

Abstract BackgroundThe Streptococcus pyogenes CRISPR system is composed of a Cas9 endonuclease (SpCas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically, SpCas9 proteins could cleave as many targeted loci as gRNAs bind in a genome.ResultsWe introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning annealed products of two oligonucleotides harboring target-binding sequence between tRNA and gRNA scaffold sequences in a pGRNA vector; and (2) assembling tRNA-gRNA units from several pGRNA vectors with a plant binary vector containing a SpCas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco (Nicotiana attenuata) protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by SpCas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites.ConclusionsThis multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.

scholarly journals A multiplex guide RNA expression system and its efficacy for plant genome engineering

2020 ◽  
Author(s):  
Youngbin Oh ◽  
Bora Lee ◽  
Hyeonjin Kim ◽  
Sang-Gyu Kim
Keyword(s):  
Genome Engineering ◽  
Binary Vector ◽  
Expression System ◽  
Plant Genome ◽  
Nicotiana Attenuata ◽  
Specific Sequence ◽  
Editing Efficiency ◽  
Guide Rna ◽  
Assembly Method ◽  
A Genome

Abstract Background: The Streptococcus pyogenes CRISPR system is composed of a Cas9 endonuclease (SpCas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically, SpCas9 proteins could cleave as many targeted loci as gRNAs bind in a genome.Results: We introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning the annealed products of two single-stranded oligonucleotide fragments harboring a complimentary target-binding sequence on each strand between tRNA and gRNA scaffold sequences in a pGRNA vector; and (2) assembling tRNA-gRNA units from several pGRNA vectors with a plant binary vector containing a SpCas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco (Nicotiana attenuata) protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by SpCas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites.Conclusions: This multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.

scholarly journals A multiplex guide RNA expression system and its efficacy for plant genome engineering

2020 ◽  
Author(s):  
Youngbin Oh ◽  
Bora Lee ◽  
Hyeonjin Kim ◽  
Sang-Gyu Kim
Keyword(s):  
Genome Engineering ◽  
Binary Vector ◽  
Expression System ◽  
Plant Genome ◽  
Nicotiana Attenuata ◽  
Specific Sequence ◽  
Editing Efficiency ◽  
Guide Rna ◽  
Assembly Method ◽  
A Genome

Abstract Background The Streptococcus pyogenes CRISPR system is composed of a Cas9 endonuclease (SpCas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically, SpCas9 proteins could cleave as many targeted loci as gRNAs bind in a genome. Results We introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning the annealed products of two single-stranded oligonucleotide fragments harboring a complimentary target-binding sequence on each strand between tRNA and gRNA scaffold sequences in a pGRNA vector; and (2) assembling tRNA-gRNA units from several pGRNA vectors with a plant binary vector containing a SpCas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco (Nicotiana attenuata) protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by SpCas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites. Conclusions This multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.

scholarly journals A multiplex guide RNA expression system and its efficacy for plant genome engineering

2020 ◽  
Author(s):  
Youngbin Oh ◽  
Bora Lee ◽  
Hyeonjin Kim ◽  
Sang-Gyu Kim
Keyword(s):  
Genome Engineering ◽  
Binary Vector ◽  
Expression System ◽  
Plant Genome ◽  
Nicotiana Attenuata ◽  
Specific Sequence ◽  
Editing Efficiency ◽  
Guide Rna ◽  
Assembly Method ◽  
A Genome

Abstract Background The Streptococcus pyogenes CRISPR system is composed of a Cas9 endonuclease (SpCas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically, SpCas9 proteins could cleave as many targeted loci as gRNAs bind in a genome. Results We introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning the annealed products of two single-stranded oligonucleotide fragments harboring a complimentary target-binding sequence on each strand between tRNA and gRNA scaffold sequences in a pGRNA vector; and (2) assembling tRNA-gRNA units from several pGRNA vectors with a plant binary vector containing a SpCas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco (Nicotiana attenuata) protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by SpCas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites. Conclusions This multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.

A Genome-Wide Analysis of the Gene Expression and Alternative Splicing Events in a Whole-Body Hypoxic Preconditioning Mouse Model

2021 ◽  
Vol 46 (5) ◽  
pp. 1101-1111
Author(s):  
Jun Li ◽  
Hongyu Zhao ◽  
Yongqiang Xing ◽  
Tongling Zhao ◽  
Lu Cai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Mouse Model ◽  
Hypoxic Preconditioning ◽  
Whole Body ◽  
Genome Wide Analysis ◽  
Genome Wide ◽  
A Genome ◽  
Alternative Splicing Events

scholarly journals Alternative splicing landscapes in Arabidopsis thaliana across tissues and stress conditions highlight major functional differences with animals

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Guiomar Martín ◽  
Yamile Márquez ◽  
Federica Mantica ◽  
Paula Duque ◽  
Manuel Irimia
Keyword(s):  
Gene Expression ◽  
Arabidopsis Thaliana ◽  
Alternative Splicing ◽  
Stress Responses ◽  
Target Genes ◽  
Intron Retention ◽  
Single Gene ◽  
Exon Skipping ◽  
Environmental Response ◽  
Biotic Stresses

Abstract Background Alternative splicing (AS) is a widespread regulatory mechanism in multicellular organisms. Numerous transcriptomic and single-gene studies in plants have investigated AS in response to specific conditions, especially environmental stress, unveiling substantial amounts of intron retention that modulate gene expression. However, a comprehensive study contrasting stress-response and tissue-specific AS patterns and directly comparing them with those of animal models is still missing. Results We generate a massive resource for Arabidopsis thaliana, PastDB, comprising AS and gene expression quantifications across tissues, development and environmental conditions, including abiotic and biotic stresses. Harmonized analysis of these datasets reveals that A. thaliana shows high levels of AS, similar to fruitflies, and that, compared to animals, disproportionately uses AS for stress responses. We identify core sets of genes regulated specifically by either AS or transcription upon stresses or among tissues, a regulatory specialization that is tightly mirrored by the genomic features of these genes. Unexpectedly, non-intron retention events, including exon skipping, are overrepresented across regulated AS sets in A. thaliana, being also largely involved in modulating gene expression through NMD and uORF inclusion. Conclusions Non-intron retention events have likely been functionally underrated in plants. AS constitutes a distinct regulatory layer controlling gene expression upon internal and external stimuli whose target genes and master regulators are hardwired at the genomic level to specifically undergo post-transcriptional regulation. Given the higher relevance of AS in the response to different stresses when compared to animals, this molecular hardwiring is likely required for a proper environmental response in A. thaliana.

scholarly journals The Arabidopsis mediator complex subunit 8 regulates oxidative stress responses

2021 ◽  
Author(s):  
Huaming He ◽  
Jordi Denecker ◽  
Katrien Van Der Kelen ◽  
Patrick Willems ◽  
Robin Pottie ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Salicylic Acid ◽  
Jasmonic Acid ◽  
Stress Responses ◽  
Negative Regulator ◽  
Mediator Complex ◽  
Defense Gene Expression ◽  
A Genome ◽  
Oxidative Stress Responses

Abstract Signaling events triggered by hydrogen peroxide (H2O2) regulate plant growth and defense by orchestrating a genome-wide transcriptional reprogramming. However, the specific mechanisms that govern H2O2-dependent gene expression are still poorly understood. Here, we identify the Arabidopsis Mediator complex subunit MED8 as a regulator of H2O2 responses. The introduction of the med8 mutation in a constitutive oxidative stress genetic background (catalase-deficient, cat2) was associated with enhanced activation of the salicylic acid pathway and accelerated cell death. Interestingly, med8 seedlings were more tolerant to oxidative stress generated by the herbicide methyl viologen (MV) and exhibited transcriptional hyperactivation of defense signaling, in particular salicylic acid- and jasmonic acid-related pathways. The med8-triggered tolerance to MV was manipulated by the introduction of secondary mutations in salicylic acid and jasmonic acid pathways. In addition, analysis of the Mediator interactome revealed interactions with components involved in mRNA processing and microRNA biogenesis, hence expanding the role of Mediator beyond transcription. Notably, MED8 interacted with the transcriptional regulator NEGATIVE ON TATA-LESS, NOT2, to control the expression of H2O2-inducible genes and stress responses. Our work establishes MED8 as a component regulating oxidative stress responses and demonstrates that it acts as a negative regulator of H2O2-driven activation of defense gene expression.

scholarly journals Development of Beet necrotic yellow vein virus ‐based vectors for multiple‐gene expression and guide RNA delivery in plant genome editing

2019 ◽  
Vol 17 (7) ◽  
pp. 1302-1315 ◽  
Author(s):  
Ning Jiang ◽  
Chao Zhang ◽  
Jun‐Ying Liu ◽  
Zhi‐Hong Guo ◽  
Zong‐Ying Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Genome Editing ◽  
Plant Genome ◽  
Yellow Vein ◽  
Multiple Gene ◽  
Guide Rna

scholarly journals CRISPR-Cas12a (Cpf1): A Versatile Tool in the Plant Genome Editing Tool Box for Agricultural Advancement

2020 ◽  
Author(s):  
Anindya Bandyopadhyay ◽  
Nagesh Kancharla ◽  
vivek javalkote ◽  
santanu dasgupta ◽  
Thomas Brutnell
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Temperature Stability ◽  
Double Strand Breaks ◽  
Plant Genome ◽  
Strand Breaks ◽  
Guide Rna ◽  
Versatile Tool ◽  
Type V ◽  
Global Population

Global population is predicted to approach 10 billion by 2050, an increase of over 2 billion from today. To meet the demands of growing, geographically and socio-economically diversified nations, we need to diversity and expand agricultural production. This expansion of agricultural productivity will need to occur under increasing biotic, and environmental constraints driven by climate change. Clustered regularly interspaced short palindromic repeats-site directed nucleases (CRISPR-SDN) and similar genome editing technologies will likely be key enablers to meet future agricultural needs. While the application of CRISPR-Cas9 mediated genome editing has led the way, the use of CRISPR-Cas12a is also increasing significantly for genome engineering of plants. The popularity of the CRISPR-Cas12a, the type V (class-II) system, is gaining momentum because of its versatility and simplified features. These include the use of a small guide RNA devoid of trans-activating crispr RNA (tracrRNA), targeting of T-rich regions of the genome where Cas9 is not suitable for use, RNA processing capability facilitating simpler multiplexing, and its ability to generate double strand breaks (DSB) with staggered ends. Many monocot and dicot species have been successfully edited using this Cas12a system and further research is ongoing to improve its efficiency in plants, including improving the temperature stability of the Cas12a enzyme, identifying new variants of Cas12a or synthetically producing Cas12a with flexible PAM sequences. In this review we provide a comparative survey of CRISPR-Cas12a and Cas9, and provide a perspective on applications of CRISPR-Cas12 in agriculture.

Abstract 124: Fetal-Like RNA Splicing Remodeling in Failing Heart Revealed by Total Transcriptome Analysis

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Chen Gao ◽  
Vincent Ren ◽  
Grace (Xinshu) Xiao ◽  
Jaunian Chen ◽  
Yibin Wang
Keyword(s):  
Alternative Splicing ◽  
Rna Splicing ◽  
Cardiac Development ◽  
Heart Diseases ◽  
Expression Profiles ◽  
Mrna Splicing ◽  
Mouse Heart ◽  
Failing Heart ◽  
Splicing Regulators ◽  
A Genome

The complexity of transcriptome and proteome is contributed by alternative splicing of mRNA. Altered mRNA splicing is also implicated in many human diseases including cancer. However, little knowledge is available about the scope of alternative splicing at whole genome level in heart diseases and even less about the mechanisms underlying the regulation of mRNA splicing in response to pathological injury in heart. Using a genome-wide RNA-Seq analysis, we have identified global alternative splicing changes associated with both development and pathological remodeling in mouse heart. Most significantly, the alternative RNA splicing events observed in failing heart mimicked the splicing profile in fetal hearts, suggesting a fetal like RNA splicing remodeling in failing hearts. After examining the expression profiles of splicing regulators in neonatal, normal adult, and failing adult hearts, Fox-1 was identified as one to be significantly down regulated in the failing and fetal hearts. Morpholino mediated Fox-1 knock-down in zebrafish embryos led to lethal phenotype associated with impaired cardiac development and function. This phenotype could be rescued by re-expressing both zebrafish and mouse Fox1 gene. Therefore, our established functional significance of Fox1 mediated RNA alternative splicing serves as a key molecular player in transcriptome remodeling during cardiac development and pathology.

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