Abstract
Modern omics platforms have made the determination of susceptible/resistance genes feasible in any species generating huge numbers of potential targets for crop protection. However, the efforts to validate these targets have been hampered by the lack of a fast, precise, and efficient gene targeting system in plants. Now, the repurposing of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has solved this problem. CRISPR/Cas9 is the latest synthetic endonuclease that has revolutionized basic research by allowing facile genome editing in prokaryotes and eukaryotes. Gene knockout is now feasible at an unprecedented efficiency with the possibility of multiplexing several targets and even genome-wide mutagenesis screening. In a short time, this powerful tool has been engineered for an array of applications beyond gene editing. Here, we briefly describe the CRISPR/Cas9 system, its recent improvements and applications in gene manipulation and single DNA/RNA molecule analysis. We summarize a few recent tests targeting plant pathogens and discuss further potential applications in pest control and plant–pathogen interactions that will inform plant breeding for crop protection.
Highlights
Genetic crosses and mutagenesis based breeding are time consuming and laborious
The clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) system has become the tool of choice for gene manipulation owing to its simplicity and the willingness of researchers to share the necessary plasmids and methods, including the various algorithms for designing guide RNA (gRNA)
Most studies focus on knocking out a single gene or a combination of a few targets, the CRISPR–Cas9 system is so powerful that it has been successfully used for genome-wide mutagenesis in mammalian (Malina et al, 2014; Peng et al, 2015) and Drosophila (Bassett et al, 2015) cells
Summary
Genetic crosses and mutagenesis based breeding are time consuming and laborious. The recent development of generation sequencing is making available fast and cost effective genomic platforms of an increasing number of species including pests, plant models and crops. CRISPR–Cas9 of Streptococcus pyogenes was engineered by replacing the first 20 nucleotides of crRNA with the intended target sequence and fusing both crRNA and tracrRNA molecules to make a single guide RNA (sgRNA) (Jinek et al, 2012). The combination of some or all of the incremental improvements in different animal and plant species could enhance gene replacement efficiency for all crops.
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