Highly specific targeted mutagenesis in plants using Staphylococcus aureus Cas9.

Hidetaka Kaya,Akira Endo,Masaki Endo,Seiichi Toki,Masafumi Mikami

doi:10.1038/srep26871

Abstract

The CRISPR/Cas9 system is an efficient and convenient tool for genome editing in plants. Cas9 nuclease derived from Streptococcus pyogenes (Sp) is commonly used in this system. Recently, Staphylococcus aureus Cas9 (SaCas9)-mediated genome editing was reported in human cells and Arabidopsis. Because SaCas9 (1053 a.a.) is smaller than SpCas9 (1368 a.a.), SaCas9 could have substantial advantages for delivering and expressing Cas9 protein, especially using virus vectors. Since the protospacer adjacent motif (PAM) sequence of SaCas9 (5′-NNGRRT-3′) differs from that of SpCas9 (5′-NGG-3′), the use of this alternative Cas9 nuclease could expand the selectivity at potential cleavage target sites of the CRISPR/Cas9 system. Here we show that SaCas9 can mutagenize target sequences in tobacco and rice with efficiencies similar to those of SpCas9. We also analyzed the base preference for ‘T’ at the 6th position of the SaCas9 PAM. Targeted mutagenesis efficiencies in target sequences with non-canonical PAMs (5′-NNGRRV-3′) were much lower than those with a canonical PAM (5′-NNGRRT-3′). The length of target sequence recognized by SaCas9 is one or two nucleotides longer than that recognized by SpCas9. Taken together, our results demonstrate that SaCas9 has higher sequence recognition capacity than SpCas9 and is useful for reducing off-target mutations in crop.

Highlights

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease (CRISPR/Cas) system, the feature widely observed in prokaryote genomes, functions as an adaptive immune system by installing short DNA fragments from viruses or plasmids into CRISPR loci using RNA from the CRISPR loci to target specific sequences in invader genomes[1,2]
We examined the patterns of mutation induced by SaCas[9] and SpCas[9] at the NtPDS gene and NtFT4 gene loci (Fig. 1b,c)
The results suggested that the ‘T’ at the 6th position of protospacer adjacent motif (PAM) is necessary in order for SaCas[9] to recognize and cleave the target sequence in plant cells

Summary

Introduction

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease (CRISPR/Cas) system, the feature widely observed in prokaryote genomes, functions as an adaptive immune system by installing short DNA fragments from viruses or plasmids into CRISPR loci using RNA from the CRISPR loci to target specific sequences in invader genomes[1,2]. The chimeric single gRNA comprises two parts: a scaffold, and a specific DNA target sequence on the 5′end. SpCas[9] mediates genome editing at a site complementary to a 20-nucleotide DNA specific sequence in the gRNA13. Several Cas[9] protein homologs have been identified and characterized for use in genome editing research[16]. Each Cas[9] protein homolog functions as an RNA-guided endonuclease, with homologs differing in their molecular weights and PAM preferences. Unlike SpCas[9], SaCas[9] requires a 21- or 22-nt DNA specific sequence and the motif 5′-NNGRRT-3′as a PAM sequence[17]. To broaden the spectrum of genome editing using the CRISPR/Cas[9] system in plants, we need to widen the choice of Cas[9] proteins beyond SpCas[9]. Our results suggest that SaCas[9] can direct highly specific genome editing activity, and will be useful for targeted genome editing in crops

Methods

Results

Conclusion