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

Youngbin Oh,Sang-Gyu Kim,Bora Lee,Hyeonjin Kim

doi:10.1186/s13007-020-00580-x

Youngbin Oh, Sang-Gyu Kim + Show 2 more

Open Access

https://doi.org/10.1186/s13007-020-00580-x

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Abstract

BackgroundThe Streptococcus pyogenes CRISPR system is composed of a Cas9 endonuclease (SpCas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically, SpCas9 proteins could cleave as many targeted loci as gRNAs bind in a genome.ResultsWe 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 SpCas9 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 SpCas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites.ConclusionsThis multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.

Highlights

The Streptococcus pyogenes CRISPR system is composed of a Cas9 endonuclease (SpCas9) and a singlestranded guide RNA harboring a target-specific sequence
The CRISPR system derived from Streptococcus pyogenes consists of a CRISPR-associated protein 9 (SpCas9) endonuclease fused with a nuclear localization signal and a single-stranded guide RNA that transfers the SpCas9 to the target locus [1,2,3,4]
New system for cloning a multiplex Pre-tRNAGly gene (tRNA)‐guide RNA (gRNA) construct To express multiple gRNAs in plants, we first developed a simple, fast and PCR-free cloning method (Fig. 1). This cloning platform consists of a pre-cloned vector, Pre-cloned vector with single gRNA (pGRNA), which carries a single unit of a tRNA-gRNA scaffold, and an acceptor vector, which harbors the SpCas9-coding sequence and a selection marker (Fig. 1c)

Summary

Introduction

The Streptococcus pyogenes CRISPR system is composed of a Cas endonuclease (SpCas9) and a singlestranded guide RNA (gRNA) harboring a target-specific sequence. When a gRNA has a 14-nt or 15-nt target binding sequence, the SpCas9-gRNA complex cannot cleave the gRNA recognition site but can bind to the target site [17, 18] Attaching this dead complex to the vicinity of the normal gRNA-binding site increases its genome editing efficacy in rice [16]. Such results suggest that SpCas9gRNA binding itself enhances the chromatin accessibility of other Cas9-gRNAs targeting the proximal site. This approach can be used widely to improve the efficiency of genome editing even without knowing complex chromatin structures

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