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.
Highlights
Genetic diversity plays a crucial role in the development of novel plant varieties
This review provides a concrete overview of the fundamentals of the CRISPR-Cas9 system and its application in plant genome editing
Despite its superior and proven ability to carry out targeted genome modifications, the application of CRISPR-Cas9 in plants is still troubled by certain limitations and regulatory concerns
Summary
Genetic diversity plays a crucial role in the development of novel plant varieties. In this regard, gene diversification for the improved genetic architecture of agricultural crops has been practiced for years via conventional plant breeding techniques or through physicochemical and biological-induced random mutagenesis [1]. (TALENs), which all rely on protein-based systems with customizable DNA-binding specificities for targeting gene sequence, and the more recent platform clustered regularly interspaced short palindromic repeats-CRISPR associated 9 nuclease (CRISPR-Cas9), which depends on RNA as a targeting moiety that directs the nuclease to a defined DNA sequence [5,6] These genome editing technologies work through employing site-directed nucleases to induce double-strand breaks (DSBs) at predefined genomic loci. In contrast to the HR-mediated repair mechanism, the NHEJ repair pathway occurs with higher frequency in most organisms, including plants [10] The ability of these genome editing technologies to induce DSB at specific genomic locus has made them a powerful tool for targeted genome modifications. The review presents the current and emerging methodological advancements and optimization approaches for simple and efficient genome editing systems
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