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.

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 Possible differences in efficiency of guide RNA with different spacer sequence in CRISPR knock-down or knock-out of particular gene

2019 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Gene Expression ◽  
Genome Editing ◽  
Target Gene ◽  
Target Genes ◽  
Fluorescent Protein ◽  
Bacterial Cells ◽  
Fluorescent Intensity ◽  
Guide Rna ◽  
Targeting Efficiency ◽  
Template Dna

Cluster regularly interspersed short palindromic repeats (CRISPR) mediated genome editing has emerged as the dominant technique for modulating the expression of target genes. Specifically, when coupled with different effectors, CRISPR could be utilized to either activate or repress gene expression. Specificity of the CRISPR gene editing method arises from the unique spacer sequence in guide RNA that mediates the specific localization of Cas9 endonuclease to particular stretches of DNA. However, complementary base pairing between the guide RNA and template DNA depends critically on existence of protospacer adjacent motif (PAM) sequence immediately downstream of the spacer sequence. Such three nucleotide PAM sequence could be present at multiple loci in a given gene, which meant that different spacer sequence could be incorporated in guide RNA design to target the same gene. Given that different spacer sequences have different binding affinities to template DNA, differences could exist in the efficiency in which CRISPR-Cas9 could be guided to generate a double strand break in a particular gene locus. Using green fluorescent protein (GFP) reporter gene expressed in recombinant Escherichia coli as experimental system, this study sought to understand if differences in targeting efficiency exist between guide RNA with different spacer sequence that could target the same gene. Fluorescent intensity of cells at the population level would serve as readout of the targeting efficiency. For example, spacer sequence in guide RNA that could better activate the endonuclease activity of Cas9 would result in lower fluorescent intensity of GFP. To check for the effect of expression mode on targeting efficiency of guide RNA, GFP gene would be expressed on a plasmid in E. coli as well as integrated into the genome of the bacterium. Doing so would provide critical information on whether the CRISPR-Cas9 system has differentiated efficacy in generating double strand breaks in genomic versus plasmid DNA. Such information would inform future experimental design involving CRISPR-Cas9 genome editing technology as well as hold implications on how CRISPR evolved as an adaptive immune system in defending bacterial cells against foreign DNA. Given the goal of the study to understand the relative extent in which a target gene would be disrupted by CRISPR-Cas9 guided by different spacer sequence on guide RNA, no repair module for the target gene would be provided. Collectively, multiple occurrence of PAM sequence in a target gene meant that different spacer sequences could be used in CRISPR-Cas9 to downregulate gene expression. Relative efficacies of different spacer sequence in guide RNA in achieving targeted gene inactivation remain poorly understood and constitutes the basis of this study, which hopefully would provide guidance on the selection of specific spacer sequence that would yield the most efficacious disruption of gene expression at the genome and plasmid level.

Ribozyme‐processed guide RNA enhances virus‐mediated plant genome editing

2021 ◽  
pp. 2100189
Author(s):  
Youngbin Oh ◽  
Hyeonjin Kim ◽  
Hyo‐Jun Lee ◽  
Sang‐Gyu Kim
Keyword(s):  
Genome Editing ◽  
Plant Genome ◽  
Guide Rna

scholarly journals Creation of a genetic vector carrying a synthesis bacterial protein gene CAS9 for plant genome editing

2020 ◽  
Vol 26 ◽  
pp. 176-182
Author(s):  
I. S. Hnatiuk ◽  
O. I. Varchenko ◽  
M. F. Parii ◽  
Yu. V. Symonenko
Keyword(s):  
Genetic Transformation ◽  
Genome Editing ◽  
Plant Genome ◽  
Bacterial Protein ◽  
Rna Sequences ◽  
Genetic Construct ◽  
Guide Rna ◽  
Cas9 Protein ◽  
Cloning Method ◽  
Genetic Constructs

Aim. To create a genetic construct carrying the bacterial protein Cas9 gene, the reporter β-glucuronidase gus gene, as well as the marker phosphinotricin-N-acetyltransferase bar gene for plant genome editing. Methods. Molecular-biological, biotechnological, microbiological and bioinformatic methods were used in the study; Golden Gate molecular cloning method was used to create genetic constructs. Results. The genetic construct pSPE2053 which carries the Cas9 endonuclease gene, the gus and bar genes was created; the assembly correctness of all vector elements was confirmed by polymerase chain reaction; the construct was transferred to Escherichia coli and Agrobacterium tumefaciens cells; β-glucuronidase gene expression was verified by histochemical analysis after Nicotiana rustica L transient genetic transformation. Conclusions. The created genetic construct can be used to edit the plant genome for both stable and transient genetic transformation to accumulate recombinant Cas9 protein. The guide RNA sequences may be subsequently transferred into such plants using either stable or transient genetic transformation or traditional crossing methods. Keywords: cloning, genetic construction, gus and bar genes, Cas9 endonuclease protein, transient expression. 

scholarly journals A geminivirus-based guide RNA delivery system for CRISPR/Cas9 mediated plant genome editing

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Kangquan Yin ◽  
Ting Han ◽  
Guang Liu ◽  
Tianyuan Chen ◽  
Ying Wang ◽  
...  
Keyword(s):  
Genome Editing ◽  
Delivery System ◽  
Plant Genome ◽  
Guide Rna

scholarly journals In silico experiments validate that twenty nucleotide spacer sequence provides precise targeting of bacterial genes in CRISPR-Cas9

2019 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Genome Editing ◽  
In Silico ◽  
Bacterial Genome ◽  
Specific Gene ◽  
Guide Rna ◽  
Theoretical Predictions ◽  
Modulation Of Gene Expression ◽  
Target Effects

As a genome editing tool useful for modulating the expression of different genes, CRISPR-Cas9 is known for its precision in targeting specific genes. To do this, CRISPR-Cas9 utilizes a guide RNA for guiding the Cas9 endonuclease to specific stretches of the DNA for genome editing or modulation of gene expression. Guide RNA comprises a spacer sequence and a protospacer adjacent motif (PAM) sequence. Both components work together to help target Cas9 to a specific stretch of DNA within a gene. In particular, spacer sequence provides a unique address for localizing Cas9 to specific stretch of DNA. But, possibility exists that there could be off-target effects for particular spacer sequence used in guide RNA. Specifically, spacer sequence might engage in complementary base pairing with other stretches of DNA in the bacterial genome, and result in additional genome editing or modulation of gene expression at genes that are not targeted. Results from an in silico experiment conducted with the rpoH gene of Pseudomonas aeruginosa PAO1 revealed that all spacer sequences derived from different stretches of the rpoH gene did not elicit off-target effects in the genome of the bacterium. This concurs with theoretical predictions that the probability of off-target effects from a 20 nucleotide long spacer region is vanishingly small. Hence, a 20 nucleotide spacer sequence in guide RNA should provide a unique DNA address for precise targeting of specific gene in the genome of a bacterium. Collectively, off-target effects of CRISPR-Cas9 is a valid concern for both genetic engineering and genome editing applications as targeting of additional genes from the desired one would result in unpredictable physiological and biochemical impacts on the cell. Using the rpoH gene of Pseudomonas aeruginosa PAO1 as example, results from an in silico experiment examining possible off-target effects of different 20 nucleotide spacer sequence able to target the sense and antisense strand of the gene revealed no off-target effects. Specifically, each spacer sequence used could only target the intended rpoH gene, which concurs with theoretical predictions of vanishingly small possibility of off-target effects on a bacterial genome from a 20 nucleotide spacer sequence. Overall, the results highlight that use of a 20 nucleotide spacer sequence in guide RNA could offer precise targeting of specific gene in a bacterium.

scholarly journals In silico experiments validate that twenty nucleotide spacer sequence provides precise targeting of bacterial genes in CRISPR-Cas9

2019 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Genome Editing ◽  
In Silico ◽  
Bacterial Genome ◽  
Specific Gene ◽  
Guide Rna ◽  
Theoretical Predictions ◽  
Modulation Of Gene Expression ◽  
Target Effects

As a genome editing tool useful for modulating the expression of different genes, CRISPR-Cas9 is known for its precision in targeting specific genes. To do this, CRISPR-Cas9 utilizes a guide RNA for guiding the Cas9 endonuclease to specific stretches of the DNA for genome editing or modulation of gene expression. Guide RNA comprises a spacer sequence and a protospacer adjacent motif (PAM) sequence. Both components work together to help target Cas9 to a specific stretch of DNA within a gene. In particular, spacer sequence provides a unique address for localizing Cas9 to specific stretch of DNA. But, possibility exists that there could be off-target effects for particular spacer sequence used in guide RNA. Specifically, spacer sequence might engage in complementary base pairing with other stretches of DNA in the bacterial genome, and result in additional genome editing or modulation of gene expression at genes that are not targeted. Results from an in silico experiment conducted with the rpoH gene of Pseudomonas aeruginosa PAO1 revealed that all spacer sequences derived from different stretches of the rpoH gene did not elicit off-target effects in the genome of the bacterium. This concurs with theoretical predictions that the probability of off-target effects from a 20 nucleotide long spacer region is vanishingly small. Hence, a 20 nucleotide spacer sequence in guide RNA should provide a unique DNA address for precise targeting of specific gene in the genome of a bacterium. Collectively, off-target effects of CRISPR-Cas9 is a valid concern for both genetic engineering and genome editing applications as targeting of additional genes from the desired one would result in unpredictable physiological and biochemical impacts on the cell. Using the rpoH gene of Pseudomonas aeruginosa PAO1 as example, results from an in silico experiment examining possible off-target effects of different 20 nucleotide spacer sequence able to target the sense and antisense strand of the gene revealed no off-target effects. Specifically, each spacer sequence used could only target the intended rpoH gene, which concurs with theoretical predictions of vanishingly small possibility of off-target effects on a bacterial genome from a 20 nucleotide spacer sequence. Overall, the results highlight that use of a 20 nucleotide spacer sequence in guide RNA could offer precise targeting of specific gene in a bacterium.

Design of Guide RNA for CRISPR/Cas Plant Genome Editing

2020 ◽  
Vol 54 (1) ◽  
pp. 24-42 ◽  
Author(s):  
G. A. Gerashchenkov ◽  
N. A. Rozhnova ◽  
B. R. Kuluev ◽  
O. Yu. Kiryanova ◽  
G. R. Gumerova ◽  
...  
Keyword(s):  
Genome Editing ◽  
Plant Genome ◽  
Guide Rna

scholarly journals Possible differences in efficiency of guide RNA with different spacer sequence in CRISPR knock-down or knock-out of particular gene

2019 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Gene Expression ◽  
Genome Editing ◽  
Target Gene ◽  
Target Genes ◽  
Fluorescent Protein ◽  
Bacterial Cells ◽  
Fluorescent Intensity ◽  
Guide Rna ◽  
Targeting Efficiency ◽  
Template Dna

Cluster regularly interspersed short palindromic repeats (CRISPR) mediated genome editing has emerged as the dominant technique for modulating the expression of target genes. Specifically, when coupled with different effectors, CRISPR could be utilized to either activate or repress gene expression. Specificity of the CRISPR gene editing method arises from the unique spacer sequence in guide RNA that mediates the specific localization of Cas9 endonuclease to particular stretches of DNA. However, complementary base pairing between the guide RNA and template DNA depends critically on existence of protospacer adjacent motif (PAM) sequence immediately downstream of the spacer sequence. Such three nucleotide PAM sequence could be present at multiple loci in a given gene, which meant that different spacer sequence could be incorporated in guide RNA design to target the same gene. Given that different spacer sequences have different binding affinities to template DNA, differences could exist in the efficiency in which CRISPR-Cas9 could be guided to generate a double strand break in a particular gene locus. Using green fluorescent protein (GFP) reporter gene expressed in recombinant Escherichia coli as experimental system, this study sought to understand if differences in targeting efficiency exist between guide RNA with different spacer sequence that could target the same gene. Fluorescent intensity of cells at the population level would serve as readout of the targeting efficiency. For example, spacer sequence in guide RNA that could better activate the endonuclease activity of Cas9 would result in lower fluorescent intensity of GFP. To check for the effect of expression mode on targeting efficiency of guide RNA, GFP gene would be expressed on a plasmid in E. coli as well as integrated into the genome of the bacterium. Doing so would provide critical information on whether the CRISPR-Cas9 system has differentiated efficacy in generating double strand breaks in genomic versus plasmid DNA. Such information would inform future experimental design involving CRISPR-Cas9 genome editing technology as well as hold implications on how CRISPR evolved as an adaptive immune system in defending bacterial cells against foreign DNA. Given the goal of the study to understand the relative extent in which a target gene would be disrupted by CRISPR-Cas9 guided by different spacer sequence on guide RNA, no repair module for the target gene would be provided. Collectively, multiple occurrence of PAM sequence in a target gene meant that different spacer sequences could be used in CRISPR-Cas9 to downregulate gene expression. Relative efficacies of different spacer sequence in guide RNA in achieving targeted gene inactivation remain poorly understood and constitutes the basis of this study, which hopefully would provide guidance on the selection of specific spacer sequence that would yield the most efficacious disruption of gene expression at the genome and plasmid level.

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