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Background  The  Streptococcus pyogenes  CRISPR system is composed of a Cas9 endonuclease ( Sp Cas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically,  Sp Cas9 proteins could cleave as many targeted loci as gRNAs bind in a genome.    Results  We introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning the annealed products of two single-stranded oligonucleotide fragments harboring a complimentary target-binding sequence on each strand between tRNA and gRNA scaffold sequences in a pGRNA vector; and (2) assembling tRNA-gRNA units from several pGRNA vectors with a plant binary vector containing a  Sp Cas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco ( Nicotiana attenuata ) protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by  Sp Cas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites.    Conclusions  This multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.

A multiplex guide RNA expression system and its efficacy for plant genome engineering