Development of CRISPR/Cas9 system for plant genome editing

Abstract

these vectors express SpCas9 proteins and/or gRNA in plants. This system is called the virus-induced genome editing (VIGE) system (Oh et al., 2021a). In tobacco rattle virus (TRV)-based VIGE system, subgenomic promoters have been used to express gRNAs. However, the transcription initiation sites of the subgenomic promoters remain elusive. Here, we examined the sequence of gRNAs expressed by subgenomic promoters and found the variable length of overhangs at 5′-end of gRNAs. The overhangs at 5′-end of gRNA decrease the cleavage activity of SpCas9. To overcome this problem, we inserted hammerhead ribozyme between the subgenomic promoter and gRNA and confirmed that gRNAs with a precise 5′-end increase the editing efficacy in Nicotiana attenuata (wild tobacco) which express the SpCas9 under the control of 35S promoter (35S:Cas9). This system will be widely used for editing target genes in plants with high efficiency (Oh et al., 2021b). Although the mutation was observed in the infected leaves (10~44%), no edited seeds were found in the M1 population of TRV-infected 35S:Cas9 plants. To increase the expression of SpCas9 in germ cells, we generated N. attenuata transgenic lines expressing SpCas9 under the control of the RPS5A promoter (pRPS5A:Cas9). The RPS5A promoter-driven SpCas9 successfully produced monoallelic mutations with virus-delivered gRNA in seeds of TRV-infected pRPS5A:Cas9. Targeted mutations are essential to understanding gene function in plants

this editing method can dramatically reduce the time and effort required to generate gene-edited plants.

The 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. We introduce a PCR-free multiple gRNA cloning system for editing plant genomes (Oh et al., 2020). 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. This multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing. To generate gene-edited plants without tissue culture, plant viral vectors have been engineered