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 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 Development of a Csy4-processed guide RNA delivery system with soybean-infecting virus ALSV for genome editing

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yanjie Luo ◽  
Ren Na ◽  
Julia S. Nowak ◽  
Yang Qiu ◽  
Qing Shi Lu ◽  
...  
Keyword(s):  
Genome Editing ◽  
Delivery System ◽  
Gene Function ◽  
Function Analysis ◽  
Soybean Plant ◽  
Apple Latent Spherical Virus ◽  
Guide Rna ◽  
Guide Rnas ◽  
Plant Trait ◽  
Trait Improvement

Abstract Background A key issue for implementation of CRISPR-Cas9 genome editing for plant trait improvement and gene function analysis is to efficiently deliver the components, including guide RNAs (gRNAs) and Cas9, into plants. Plant virus-based gRNA delivery strategy has proven to be an important tool for genome editing. However, its application in soybean which is an important crop has not been reported yet. ALSV (apple latent spherical virus) is highly infectious virus and could be explored for delivering elements for genome editing. Results To develop a ALSV-based gRNA delivery system, the Cas9-based Csy4-processed ALSV Carry (CCAC) system was developed. In this system, we engineered the soybean-infecting ALSV to carry and deliver gRNA(s). The endoribonuclease Csy4 effectively releases gRNAs that function efficiently in Cas9-mediated genome editing. Genome editing of endogenous phytoene desaturase (PDS) loci and exogenous 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) sequence in Nicotiana. benthamiana (N. benthamiana) through CCAC was confirmed using Sanger sequencing. Furthermore, CCAC-induced mutagenesis in two soybean endogenous GW2 paralogs was detected. Conclusions With the aid of the CCAC system, the target-specific gRNA(s) can be easily manipulated and efficiently delivered into soybean plant cells by viral infection. This is the first virus-based gRNA delivery system for soybean for genome editing and can be used for gene function study and trait improvement.

scholarly journals A viral guide RNA delivery system for CRISPR-based transcriptional activation and heritable targeted DNA demethylation in Arabidopsis thaliana

2020 ◽  
Author(s):  
Basudev Ghoshal ◽  
Brandon Vong ◽  
Colette L. Picard ◽  
Feng Suhua ◽  
Janet May Tam ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Genome Editing ◽  
Delivery System ◽  
Transcriptional Activation ◽  
Rna Viruses ◽  
Tobacco Rattle Virus ◽  
Dna Demethylation ◽  
Guide Rna ◽  
Epigenome Editing ◽  
Guide Rnas

AbstractPlant RNA viruses are used as delivery vectors for their high level of accumulation and efficient spread during virus multiplication and movement. Utilizing this concept, several viral-based guide RNA delivery platforms for CRISPR-Cas9 genome editing have been developed. The CRISPR-Cas9 system has also been adapted for epigenome editing. While systems have been developed for CRISPR-Cas9 based gene activation or site-specific DNA demethylation, viral delivery of guide RNAs remains to be developed for these purposes. To address this gap we have developed a tobacco rattle virus (TRV)-based single guide RNA delivery system for epigenome editing in Arabidopsis thaliana. Because tRNA-like sequences have been shown to facilitate the cell-to-cell movement of RNAs in plants, we used the tRNA-guide RNA expression system to express guide RNAs from the viral genome to promote heritable epigenome editing. We demonstrate that the tRNA-gRNA system with TRV can be used for both transcriptional activation and targeted DNA demethylation in Arabidopsis. We achieved up to ~8% heritability of the induced demethylation phenotype in the progeny of virus inoculated plants. We did not detect the virus in the next generation, indicating effective clearance of the virus from plant tissues. Thus, TRV delivery, combined with a specific tRNA-gRNA architecture, provides for fast and effective epigenome editing.Author summaryThe discovery of CRISPR-CAS9 and its non-catalytic variants have provided enormous capacity for crop improvement and basic research by modifying the genome and the epigenome. The standard methods for delivering genome and epigenome editing reagents to plants consist of generating stable transgenic lines through tissue culture processes, which have several drawbacks including the need for plant regeneration and crossing. To overcome some of these challenges, plant virus-based platforms are being developed for genome editing. Although viruses have a limited cargo capacity, limiting the use of viruses to encode entire editing systems, guide RNAs have been successfully delivered to transgenic CAS9 expressing plants for genome editing. However, the use of viruses for CRISPR-based epigenome editing and transcriptional activation have not yet been explored. In this study we show that viral delivery of guide RNAs using a modified tobacco rattle virus can be used for transcriptional activation and heritable epigenome editing. This study advances the use of plant RNA viruses as delivery agents for epigenome editing.

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. 

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

An Era of CRISPR/ Cas9 Mediated Plant Genome Editing

2018 ◽  
pp. 47-54 ◽  
Author(s):  
Haris Khurshid ◽  
Sohail Ahmad Jan ◽  
Zabta Khan Shinwari ◽  
Muhammad Jamal ◽  
Sabir Hussain Shah
Keyword(s):  
Genome Editing ◽  
Plant Genome

scholarly journals Exosome/Liposome-like Nanoparticles: New Carriers for CRISPR Genome Editing in Plants

2021 ◽  
Vol 22 (14) ◽  
pp. 7456
Author(s):  
Mousa A. Alghuthaymi ◽  
Aftab Ahmad ◽  
Zulqurnain Khan ◽  
Sultan Habibullah Khan ◽  
Farah K. Ahmed ◽  
...  
Keyword(s):  
Genome Editing ◽  
Viral Vectors ◽  
Polymeric Materials ◽  
Inorganic Nanoparticles ◽  
Plant Genome ◽  
Delivery Methods ◽  
Detailed Consideration ◽  
Crispr System ◽  
Efficient Delivery ◽  
Low Cytotoxicity

Rapid developments in the field of plant genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems necessitate more detailed consideration of the delivery of the CRISPR system into plants. Successful and safe editing of plant genomes is partly based on efficient delivery of the CRISPR system. Along with the use of plasmids and viral vectors as cargo material for genome editing, non-viral vectors have also been considered for delivery purposes. These non-viral vectors can be made of a variety of materials, including inorganic nanoparticles, carbon nanotubes, liposomes, and protein- and peptide-based nanoparticles, as well as nanoscale polymeric materials. They have a decreased immune response, an advantage over viral vectors, and offer additional flexibility in their design, allowing them to be functionalized and targeted to specific sites in a biological system with low cytotoxicity. This review is dedicated to describing the delivery methods of CRISPR system into plants with emphasis on the use of non-viral vectors.

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