scholarly journals Efficient Genome Editing Using CRISPR/Cas9 Technology in Chicory

2019 ◽  
Vol 20 (5) ◽  
pp. 1155 ◽  
Author(s):  
Guillaume Bernard ◽  
David Gagneul ◽  
Harmony Alves Dos Santos ◽  
Audrey Etienne ◽  
Jean-Louis Hilbert ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mutation Frequency ◽  
Function Analysis ◽  
Expression System ◽  
Crop Improvement ◽  
Targeted Mutagenesis ◽  
Plant Genome ◽  
A Genome ◽  
Transformation Methods ◽  
Biallelic Mutations

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated with protein CAS9) is a genome-editing tool that has been extensively used in the last five years because of its novelty, affordability, and feasibility. This technology has been developed in many plant species for gene function analysis and crop improvement but has never been used in chicory (Cichorium intybus L.). In this study, we successfully applied CRISPR/Cas9-mediated targeted mutagenesis to chicory using Agrobacterium rhizogenes-mediated transformation and protoplast transfection methods. A U6 promoter (CiU6-1p) among eight predicted U6 promoters in chicory was selected to drive sgRNA expression. A binary vector designed to induce targeted mutations in the fifth exon of the chicory phytoene desaturase gene (CiPDS) was then constructed and used to transform chicory. The mutation frequency was 4.5% with the protoplast transient expression system and 31.25% with A. rhizogenes-mediated stable transformation. Biallelic mutations were detected in all the mutant plants. The use of A. rhizogenes-mediated transformation seems preferable as the regeneration of plants is faster and the mutation frequency was shown to be higher. With both transformation methods, foreign DNA was integrated in the plant genome. Hence, selection of vector (transgene)-free segregants is required. Our results showed that genome editing with CRISPR/Cas9 system can be efficiently used with chicory, which should facilitate and accelerate genetic improvement and functional biology.

scholarly journals Modern Trends in Plant Genome Editing: An Inclusive Review of the CRISPR/Cas9 Toolbox

2019 ◽  
Vol 20 (16) ◽  
pp. 4045 ◽  
Author(s):  
Ali Razzaq ◽  
Fozia Saleem ◽  
Mehak Kanwal ◽  
Ghulam Mustafa ◽  
Sumaira Yousaf ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Crop Improvement ◽  
Crop Plants ◽  
Targeted Mutagenesis ◽  
Plant Genome ◽  
Zinc Finger Nucleases ◽  
Base Substitution ◽  
Transcription Activator ◽  
Abiotic Stressors

Increasing agricultural productivity via modern breeding strategies is of prime interest to attain global food security. An array of biotic and abiotic stressors affect productivity as well as the quality of crop plants, and it is a primary need to develop crops with improved adaptability, high productivity, and resilience against these biotic/abiotic stressors. Conventional approaches to genetic engineering involve tedious procedures. State-of-the-art OMICS approaches reinforced with next-generation sequencing and the latest developments in genome editing tools have paved the way for targeted mutagenesis, opening new horizons for precise genome engineering. Various genome editing tools such as transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and meganucleases (MNs) have enabled plant scientists to manipulate desired genes in crop plants. However, these approaches are expensive and laborious involving complex procedures for successful editing. Conversely, CRISPR/Cas9 is an entrancing, easy-to-design, cost-effective, and versatile tool for precise and efficient plant genome editing. In recent years, the CRISPR/Cas9 system has emerged as a powerful tool for targeted mutagenesis, including single base substitution, multiplex gene editing, gene knockouts, and regulation of gene transcription in plants. Thus, CRISPR/Cas9-based genome editing has demonstrated great potential for crop improvement but regulation of genome-edited crops is still in its infancy. Here, we extensively reviewed the availability of CRISPR/Cas9 genome editing tools for plant biotechnologists to target desired genes and its vast applications in crop breeding research.

scholarly journals Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system

Plant Methods ◽  
2013 ◽  
Vol 9 (1) ◽  
pp. 39 ◽  
Author(s):  
Khaoula Belhaj ◽  
Angela Chaparro-Garcia ◽  
Sophien Kamoun ◽  
Vladimir Nekrasov
Keyword(s):  
Genome Editing ◽  
Crop Plants ◽  
Targeted Mutagenesis ◽  
Plant Genome

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 230 Genome editing applications in plants: high-throughput CRISPR/Cas editing for crop improvement

2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 56-56
Author(s):  
Michael Thomson
Keyword(s):  
Genome Editing ◽  
High Throughput ◽  
Positional Cloning ◽  
Gene Editing ◽  
Crop Improvement ◽  
Cost Effective ◽  
Gene Families ◽  
Ease Of Use ◽  
Plant Genome ◽  
Wide Range

Abstract The precision and ease of use of CRISPR nucleases, such as Cas9 and Cpf1, for plant genome editing has the potential to accelerate a wide range of applications for crop improvement. For upstream research on gene discovery and validation, rapid gene knock-outs can enable testing of single genes and multi-gene families for functional effects. Large chromosomal deletions can test the function of tandem gene arrays and assist with positional cloning of QTLs by helping to narrow down the target region. Nuclease-deactivated Cas9 fusion proteins with transcriptional activators and repressors can be used to up and down-regulate gene expression. Even more promising, gene insertions and allele replacements can provide the opportunity to rapidly test the effects of different alleles at key loci in the same genetic background, providing a more precise alternative to marker-assisted backcrossing. Recently, Texas A&M AgriLife Research has supported the development of a Crop Genome Editing Lab at Texas A&M working towards optimizing a high-throughput gene editing pipeline and providing an efficient and cost-effective gene editing service for research and breeding groups. The lab is using rice as a model to test and optimize new approaches aimed towards overcoming current bottlenecks. For example, a wealth of genomics data from the rice community enables the development of novel approaches to predict which genes and target modifications may be most beneficial for crop improvement, taking advantage of known major genes, high-resolution GWAS data, multiple high-quality reference genomes, transcriptomics data, and resequencing data from the 3,000 Rice Genomes Project. Current projects have now expanded to work across multiple crops to provide breeding and research groups with a rapid gene editing pipeline to test candidate genes in their programs, with the ultimate goal of developing nutritious, high-yielding, stress-tolerant crops for the future.

scholarly journals Generating novel plant genetic variation via genome editing to escape the breeding lottery

2021 ◽  
Author(s):  
Nathaniel Schleif ◽  
Shawn M. Kaeppler ◽  
Heidi F. Kaeppler
Keyword(s):  
Genetic Variation ◽  
Plant Breeding ◽  
Genome Editing ◽  
Breeding Success ◽  
Crop Improvement ◽  
Random Mutagenesis ◽  
Plant Genome ◽  
Gene Pools ◽  
Plant Genotype ◽  
Wild Accessions

AbstractPlant breeding relies on the presence of genetic variation, which is generated by a random process of mutagenesis that acts on existing gene pools. This variation is then recombined into new forms at frequencies impacted by the local euchromatin and heterochromatin environment. The result is a genetic lottery where plant breeders face increasingly low odds of generating a “winning” plant genotype. Genome editing tools enable targeted manipulation of the genome, providing a means to increase genetic variation and enhancing the chances for plant breeding success. Editing can be applied in a targeted way, where known genetic variation that improves performance can be directly brought into lines of interest through either deletion or insertion. This empowers approaches that are traditionally difficult such as novel domestication and introgression of wild accessions into a germplasm pool. Furthermore, broader editing-mediated approaches such as recombination enhancement and targeted random mutagenesis bring novel ways of variation creation to the plant breeding toolbox. Continued development and application of plant genome editing tools will be needed to aid in meeting critical global crop improvement needs.

scholarly journals CRISPR/Cas9-Mediated Targeted Mutagenesis of Wild Soybean (Glycine soja) Hairy Roots Altered the Transcription Profile of the Mutant

2020 ◽  
Vol 12 (9) ◽  
pp. 14
Author(s):  
Fengjuan Niu ◽  
Qiyan Jiang ◽  
Rui Cheng ◽  
Xianjun Sun ◽  
Zheng Hu ◽  
...  
Keyword(s):  
Glycine Max ◽  
Genome Editing ◽  
Hairy Roots ◽  
Glycine Soja ◽  
Differentially Expressed Gene ◽  
Wild Soybean ◽  
Targeted Mutagenesis ◽  
Functional Identification ◽  
Transcription Profiles ◽  
Biallelic Mutations

Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated protein 9 (CRISPR/Cas9) system has been regularly applied for genome editing and gene function identification in wild soybean (Glycine max) cultivars. However, till date no studies have demonstrated successful mutagenesis in wild soybean (Glycine soja) which is the ancestor of Glycine max and rich in stress tolerance genes. In the current study, we report the successful creation of mutations in the loci encoding plasma membrane Na+/H+ antiporter (SOS1) and nonselective cation channels (NSCC) in wild soybean hairy roots using the CRISPR/Cas9 system. Two genes, GsSOS1 and GsNSCC, were mutagenized with frequencies of 28.5% and 39.9%, respectively. Biallelic mutations in GsSOS1 were detected in transgenic hairy roots. GsSOS1 mutants exhibited altered Na+/K+ ratios in the roots under both control and salt-treated conditions. However, no significant effects of GsNSCC mutation on Na+/K+ ratios were observed. RNA-Seq analysis revealed that both GsSOS1 and GsNSCC mutation altered the transcription profiles in mutant roots. Many differentially expressed gene sets that are associated with various cellular functions were identified. Our results demonstrated that CRISPR/Cas9 systems as powerful tools for wild soybean genome editing and would significantly advance the gene mining and functional identification in wild soybean.

Engineering Resistance Against Viruses in Field Crops Using CRISPR-Cas9

2021 ◽  
Vol 22 ◽  
Author(s):  
Vidya R. Hinge ◽  
Rahul L. Chavhan ◽  
Sandeep P. Kale ◽  
Penna Suprasanna ◽  
Ulhas S. Kadam
Keyword(s):  
Genome Editing ◽  
Host Resistance ◽  
Virus Resistance ◽  
Plant Virus ◽  
Crop Improvement ◽  
Plant Viruses ◽  
Plant Genome ◽  
Change Scenario ◽  
Field Crops ◽  
Plant Virus Resistance

: Various types biotic stresses affect growth and production of agricultural crops, among them viruses are of most concern which cause yield losses in all field crops; challenging global food security. Enhancement of host resistance against plant viruses is a priority for effective management of plant viral diseases. In the present context of climate change scenario, plant viruses are rapidly evolving and defeating the host resistance. Advances in genome editing techniques such as CRISPR-Cas9 [clustered regularly interspaced palindromic repeats-CRISPR-associated 9] have been recognized as a promising tool for the development of plant virus resistance. CRISPR-Cas9 genome editing tool is widely preferred due to high target specificity, simple, efficient, and reproducible genetic manipulation. CRISPR-Cas9 based virus resistance in plants has been successfully achieved through gene targeting and cleaving viral genome or altering the plant genome to enhance plant innate immunity. In this article, we have outlined the CRISPR-Cas9 system, plant immunity against viruses and use of CRISPR-Cas9 system to engineer virus resistance in plants. We also discuss prospects and challenges on the use of CRISPR-Cas9-mediated plant virus resistance in crop improvement.

scholarly journals I-SceI Endonuclease-Mediated Plant Genome Editing by Protein Transport through a Bacterial Type III Secretion System

Plants ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1070
Author(s):  
Yuki Yanagawa ◽  
Kasumi Takeuchi ◽  
Masaki Endo ◽  
Ayako Furutani ◽  
Hirokazu Ochiai ◽  
...  
Keyword(s):  
Genome Editing ◽  
Type Iii Secretion ◽  
Tobacco Plant ◽  
Plant Cells ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Plant Genome ◽  
Recognition Sequence ◽  
Type Iii ◽  
A Genome

Xanthomonas campestris is one of bacteria carrying a type III secretion system which transports their effector proteins into host plant cells to disturb host defense system for their infection. To establish a genome editing system without introducing any foreign gene, we attempted to introduce genome editing enzymes through the type III secretion system. In a test of protein transfer, X. campestris pv. campestris (Xcc) transported a considerable amount of a reporter protein sGFP-CyaA into tobacco plant cells under the control of the type III secretion system while maintaining cell viability. For proof of concept for genome editing, we used a reporter tobacco plant containing a luciferase (LUC) gene interrupted by a meganuclease I-SceI recognition sequence; this plant exhibits chemiluminescence of LUC only when a frameshift mutation is introduced at the I-SceI recognition site. Luciferase signal was observed in tobacco leaves infected by Xcc carrying an I-SceI gene which secretes I-SceI protein through the type III system, but not leaves infected by Xcc carrying a vector control. Genome-edited tobacco plant could be regenerated from a piece of infected leaf piece by repeated selection of LUC positive calli. Sequence analysis revealed that the regenerated tobacco plant possessed a base deletion in the I-SceI recognition sequence that activated the LUC gene, indicating genome editing by I-SceI protein transferred through the type III secretion system of Xcc.

scholarly journals Genome editing in plants using CRISPR type I-D nuclease

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Keishi Osakabe ◽  
Naoki Wada ◽  
Tomoko Miyaji ◽  
Emi Murakami ◽  
Kazuya Marui ◽  
...  
Keyword(s):  
Genome Editing ◽  
First Generation ◽  
Genome Engineering ◽  
Targeted Mutagenesis ◽  
Plant Genome ◽  
Type I ◽  
Crispr System ◽  
Target Dna ◽  
Short Indels

Abstract Genome editing in plants has advanced greatly by applying the clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas system, especially CRISPR-Cas9. However, CRISPR type I—the most abundant CRISPR system in bacteria—has not been exploited for plant genome modification. In type I CRISPR-Cas systems, e.g., type I-E, Cas3 nucleases degrade the target DNA in mammals. Here, we present a type I-D (TiD) CRISPR-Cas genome editing system in plants. TiD lacks the Cas3 nuclease domain; instead, Cas10d is the functional nuclease in vivo. TiD was active in targeted mutagenesis of tomato genomic DNA. The mutations generated by TiD differed from those of CRISPR/Cas9; both bi-directional long-range deletions and short indels mutations were detected in tomato cells. Furthermore, TiD can be used to efficiently generate bi-allelic mutant plants in the first generation. These findings indicate that TiD is a unique CRISPR system that can be used for genome engineering in plants.

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