scholarly journals Utilizing CRISPR-Cas in Tropical Crop Improvement: A Decision Process for Fitting Genome Engineering to Your Species

2021 ◽  
Vol 12 ◽  
Author(s):  
Kathleen A. Joo ◽  
Michael G. Muszynski ◽  
Michael B. Kantar ◽  
Ming-Li Wang ◽  
Xiaoling He ◽  
...  
Keyword(s):  
Gene Editing ◽  
Genome Engineering ◽  
Crop Improvement ◽  
Screening Methods ◽  
Decision Points ◽  
Trait Improvement ◽  
Tropical Crop ◽  
Transformation Methods ◽  
Cas Gene

Adopting modern gene-editing technologies for trait improvement in agriculture requires important workflow developments, yet these developments are not often discussed. Using tropical crop systems as a case study, we describe a workflow broken down into discrete processes with specific steps and decision points that allow for the practical application of the CRISPR-Cas gene editing platform in a crop of interest. While we present the steps of developing genome-edited plants as sequential, in practice parts can be done in parallel, which are discussed in this perspective. The main processes include 1) understanding the genetic basis of the trait along with having the crop’s genome sequence, 2) testing and optimization of the editing reagents, development of efficient 3) tissue culture and 4) transformation methods, and 5) screening methods to identify edited events with commercial potential. Our goal in this perspective is to help any lab that wishes to implement this powerful, easy-to-use tool in their pipeline, thus aiming to democratize the technology.

scholarly journals Current Advancements and Limitations of Gene Editing in Orphan Crops

2021 ◽  
Vol 12 ◽  
Author(s):  
Matthew Venezia ◽  
Kate M. Creasey Krainer
Keyword(s):  
Gene Editing ◽  
Crop Improvement ◽  
Screening Methods ◽  
In Planta ◽  
Base Editing ◽  
Guide Rna ◽  
Orphan Crops ◽  
Associated Proteins ◽  
Improvement Programs ◽  
Novel Alleles

Gene editing provides precise, heritable genome mutagenesis without permanent transgenesis, and has been widely demonstrated and applied in planta. In the past decade, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) has revolutionized the application of gene editing in crops, with mechanistic advances expanding its potential, including prime editing and base editing. To date, CRISPR/Cas has been utilized in over a dozen orphan crops with diverse genetic backgrounds, leading to novel alleles and beneficial phenotypes for breeders, growers, and consumers. In conjunction with the adoption of science-based regulatory practices, there is potential for CRISPR/Cas-mediated gene editing in orphan crop improvement programs to solve a plethora of agricultural problems, especially impacting developing countries. Genome sequencing has progressed, becoming more affordable and applicable to orphan crops. Open-access resources allow for target gene identification and guide RNA (gRNA) design and evaluation, with modular cloning systems and enzyme screening methods providing experimental feasibility. While the genomic and mechanistic limitations are being overcome, crop transformation and regeneration continue to be the bottleneck for gene editing applications. International collaboration between all stakeholders involved in crop improvement is vital to provide equitable access and bridge the scientific gap between the world’s most economically important crops and the most under-researched crops. This review describes the mechanisms and workflow of CRISPR/Cas in planta and addresses the challenges, current applications, and future prospects in orphan crops.

scholarly journals LA EDICIÓN DE GENOMAS: PLANTAS A LA CARTA

2020 ◽  
Author(s):  
Concha Gómez-Mena
Keyword(s):  
Gene Editing ◽  
Crop Improvement ◽  
Genome Sequences ◽  
New Approach ◽  
Real Possibility ◽  
On Demand ◽  
Gene Functions ◽  
History Of ◽  
The One ◽  
Cas Gene

The plants we eat are the outcome of a humans’ long history of domestication of wild species. The introduction of CRISPR/Cas gene-editing technology has provided a new approach to crop improvement and offers an interesting range of possibilities for obtaining varieties with new and healthier characteristics. The technology is based on two fundamental pillars: on the one hand, knowing complete genome sequences, and on the other, identifying gene functions. In less than a decade, the prospect of being able to design plants on demand is now no longer a dream, but a real possibility.

scholarly journals Plant viral vectors: Expanding the Possibilities of Precise Gene Editing in Plant Genomes

2021 ◽  
Author(s):  
Stuti Kujur ◽  
Muthappa Senthil-Kumar ◽  
Rahul Kumar
Keyword(s):  
Genome Editing ◽  
Plant Transformation ◽  
Viral Vectors ◽  
Gene Editing ◽  
Genome Engineering ◽  
Plant Cells ◽  
In Planta ◽  
Plant Genomes ◽  
Efficient Gene ◽  
Transformation Methods

Abstract The lack of a highly efficient method for delivering reagents for genome engineering to plant cells remains a bottleneck in achieving efficient gene-editing in plant genomes. A suite of recent reports uncovers the newly emerged roles of viral vectors, which can introduce gene-edits in plants with high mutation frequencies through in planta delivery. Here, we focus on the emerging protocols that utilized different approaches for virus-mediated genome editing in model plants. Testing of these protocols and the newly identified hypercompact Casɸ systems is needed to broaden the scope of genome-editing in most plant species, including crops, with minimized reliance on conventional plant transformation methods in the future.

scholarly journals Genotyping strategies for detecting CRISPR mutations in polyploid species: a case study-based approach in hexaploid wheat

2021 ◽  
Author(s):  
Ajay Gupta ◽  
Wanlong Li
Keyword(s):  
Genome Engineering ◽  
Crop Improvement ◽  
Cost Effective ◽  
Significant Advance ◽  
Polyploid Species ◽  
Small Indels ◽  
Cleavage Assay ◽  
Versatile Tool ◽  
Mismatch Cleavage

As a versatile tool for genome engineering, CRISPR-Cas9 has been revolutionizing the field of molecular biology, biotechnology, and crop improvement. By precisely targeting pre-selected genomic sites, CRISPR-Cas9 primarily induces insertions or deletions (indels) of variable size. Despite the significant advance in the technology per se, detecting these indels is the major and difficult part of the CRISPR program in polyploid species, like wheat, with relatively low mutation rates. A plethora of methods are available for detecting mutations, but no method is perfect for all mutation types. In this case study, we demonstrated a new, protocol for capturing length polymorphism from small indels using a nested PCR approach. This new method is tractable, efficient, and cost-effective in detecting and genotyping indels >3-bp. We also discussed the major genotyping platforms used in our wheat CRISPR projects, such as mismatch cleavage assay, restriction enzyme assay, ribonucleoprotein assay, and Sanger sequencing, for their advantages and pitfalls in wheat CRISPR mutation detection.

scholarly journals Improved Transformation and Regeneration of Indica Rice: Disruption of SUB1A as a Test Case via CRISPR-Cas9

2021 ◽  
Vol 22 (13) ◽  
pp. 6989
Author(s):  
Yuya Liang ◽  
Sudip Biswas ◽  
Backki Kim ◽  
Julia Bailey-Serres ◽  
Endang M. Septiningsih
Keyword(s):  
Gene Editing ◽  
Indica Rice ◽  
Crop Improvement ◽  
Immature Embryo ◽  
Test Case ◽  
Submergence Tolerance ◽  
Embryogenic Calli ◽  
Submergence Stress ◽  
Efficient Vector ◽  
Vector Delivery

Gene editing by use of clustered regularly interspaced short palindromic repeats (CRISPR) has become a powerful tool for crop improvement. However, a common bottleneck in the application of this approach to grain crops, including rice (Oryza sativa), is efficient vector delivery and calli regeneration, which can be hampered by genotype-dependent requirements for plant regeneration. Here, methods for Agrobacterium-mediated and biolistic transformation and regeneration of indica rice were optimized using CRISPR-Cas9 gene-editing of the submergence tolerance regulator SUBMERGENCE 1A-1 gene of the cultivar Ciherang-Sub1. Callus induction and plantlet regeneration methods were optimized for embryogenic calli derived from immature embryos and mature seed-derived calli. Optimized regeneration (95%) and maximal editing efficiency (100%) were obtained from the immature embryo-derived calli. Phenotyping of T1 seeds derived from the edited T0 plants under submergence stress demonstrated inferior phenotype compared to their controls, which phenotypically validates the disruption of SUB1A-1 function. The methods pave the way for rapid CRISPR-Cas9 gene editing of recalcitrant indica rice cultivars.

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