An Optimized RNA-Guided Cas9 System for Efficient Simplex and Multiplex Genome Editing in Barley (Hordeum vulgare L.)
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The ability to create genetic mutants is crucial for genomics studies in plants and for crop improvement. However, until recently, knockout mutants were created mainly by random mutagenesis, which produced many undesirable mutations and genomic rearrangements. With the emergence of programmable site-specific nucleases, mutations can be introduced precisely at specific genomic locations. CRISPR-Cas9 technology represents the most versatile and efficient genome editing tool to date, and the number of species in which this technology has been applied is still rising. Recently, many efforts have been made to adapt the CRISPR-Cas9 system for efficient genome editing of economically important crop species. At present, CRISPR-Cas9-based targeted mutagenesis of single and multiple target genes can be utilized in barley with near 90% efficiency. Cas9 and sgRNA can be introduced to barley plants in a single T-DNA construct via stable Agrobacterium-mediated transformation of immature embryos. Mutations induced at the target loci are detected in T0 plants, and homozygous transgene-free mutants segregate in the T1 and T2 generations. Here, we present methods and strategies for the induction of single and multiple knockout mutations in barley. The protocol covers all important steps, from target sequence selection and construct optimization and assembly to mutation detection and selection of nontransgenic mutant lines.
Key wordsBarley Cas9 sgRNA PTG Mutant Mutation detection
This research was supported by the Polish National Research Center grant 2015/17/D/NZ9/02020.
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