CRISPR-Cas9 System for Plant Genome Editing: Current Approaches and Emerging Developments

Abstract

Targeted genome editing using CRISPR-Cas9 has been widely adopted as a genetic engineering tool in various biological systems. This editing technology has been in the limelight due to its simplicity and versatility compared to other previously known genome editing platforms. Several modifications of this editing system have been established for adoption in a variety of plants, as well as for its improved efficiency and portability, bringing new opportunities for the development of transgene-free improved varieties of economically important crops. This review presents an overview of CRISPR-Cas9 and its application in plant genome editing. A catalog of the current and emerging approaches for the implementation of the system in plants is also presented with details on the existing gaps and limitations. Strategies for the establishment of the CRISPR-Cas9 molecular construct such as the selection of sgRNAs, PAM compatibility, choice of promoters, vector architecture, and multiplexing approaches are emphasized. Progress in the delivery and transgene detection methods, together with optimization approaches for improved on-target efficiency are also detailed in this review. The information laid out here will provide options useful for the effective and efficient exploitation of the system for plant genome editing and will serve as a baseline for further developments of the system. Future combinations and fine-tuning of the known parameters or factors that contribute to the editing efficiency, fidelity, and portability of CRISPR-Cas9 will indeed open avenues for new technological advancements of the system for targeted gene editing in plants.

CRISPR-Based Genome Editing Tools: Insights into Technological Breakthroughs and Future Challenges

“…The most important and challenging step after the successful delivery of CRISPR/cas9 cassette is the (a) selection of transformants with mutations and (b) the elimination of CRISPR–cas9 cassette, as the presence of it in transformations can increase the rate of off targeting [ 126 , 145 , 146 ]. However, by selfing and back crossing CRISPR-cas9 cassette can be eliminated in T 2 generation and Cas9 free mutants can be produced.…”

RNA Interference and CRISPR/Cas Gene Editing for Crop Improvement: Paradigm Shift towards Sustainable Agriculture

“…The selection of the promoter for the expression of Cas9 and sgRNAs and the configuration of these expression cassettes are crucial to achieve proficient genome editing. Commonly, there are three expression strategies for the CRISPR-Cas system in plants, i.e., a mixed dual promoter system, a dual Pol II promoter system, and a single transcriptional unit system (STU) [232]. Although the STU system is considered superior over other two systems, it has limitations i.e., refinement of the system, nonoptimal expression system, and difficult post-transcriptional processes [232].…”

“…Commonly, there are three expression strategies for the CRISPR-Cas system in plants, i.e., a mixed dual promoter system, a dual Pol II promoter system, and a single transcriptional unit system (STU) [232]. Although the STU system is considered superior over other two systems, it has limitations i.e., refinement of the system, nonoptimal expression system, and difficult post-transcriptional processes [232]. The limitations could be combatted by using a bidirectional promoter system that can initiate transcription in both orientations [233].…”

Induced Genetic Variations in Fruit Trees Using New Breeding Tools: Food Security and Climate Resilience

“…These may include a low mutation efficiency/rate, unwanted (off-target) mutations, inefficient gene delivery techniques, in vitro regeneration dependency, persistent activity in subsequent generations, the spread of transgene to other plants and reversion of mutation via cross-pollination, difficulties of execution in woody plants and long-life cycle, genotypic chimerism and strict GMO regulations. Most of these issues can be tackled through a meticulous approach by avoiding erroneous gRNA design, choosing the best Cas protein variant, designing a better expression cassette (including a promoter), employing a highly efficient DNA delivery approach and targeting the right tissue types [ 245 , 246 , 247 ].…”