Genome editing for crop improvement: Status and prospects

Abstract

Genome editing using sequence-specific nucleases has been one of the fast-evolving technologies ever since the discovery of CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated endonuclease) system. CRISPR locus present in the genomes of various bacteria and archaea offer a programmable defense mechanism against foreign agents like viruses. The understanding of this biological phenomenon leads to the development of genome editing tool for making specific modifications in the genomes of various organisms. The CRISPR/Cas9 technology basically depends upon two components: Cas9 enzyme and sgRNA (single-guide RNA). These two components are delivered into plant cells using different gene transfer methods such as Agrobacterium-mediated, biolistic (particle gun) approach, pre-assembled ribonucleoproteins followed by homology-based detection of target DNA by sgRNA, DNA cleavage by Cas9, and DNA repair by native cell machinery employing nonhomologous end joining. This usually leads to a frameshift and knockout mutation in the targeted gene. Providing a template DNA for homology-dependent repair can extend this technique to knock in mutations as well. This technology thus opens up a unique opportunity for directed alterations in chosen genes. Hence, CRISPR/Cas technology has huge potential as a precise and rapid plant breeding method. The absence of foreign DNA introduction (particularly in the case of gene knockout) is anticipated to attract fewer biosafety concerns as compared to GMOs in the regulatory frameworks coming up in different countries. This chapter reviews the CRISPR-Cas strategy by focusing on components of the tool kit and available variants, delivery into plant cells, and gene modification detection assays. A set of crop improvement-related studies, targeting genes of basic as well as applied significance, are listed to illustrate its current use.