Highly efficient heritable plant genome engineering using Cas9 orthologues from Streptococcus thermophilus and Staphylococcus aureus

Abstract

SUMMARYThe application of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system of Streptococcus pyogenes (SpCas9) is currently revolutionizing genome engineering in plants. However, synthetic plant biology will require more complex manipulations of genomes and transcriptomes. The simultaneous addressing of different specific genomic sites with independent enzyme activities within the same cell is a key to this issue. Such approaches can be achieved by the adaptation of additional bacterial orthologues of the CRISPR/Cas system for use in plant cells. Here, we show that codon-optimised Cas9 orthologues from Streptococcus thermophilus (St1Cas9) and Staphylococcus aureus (SaCas9) can both be used to induce error-prone non-homologous end-joining-mediated targeted mutagenesis in the model plant Arabidopsis thaliana at frequencies at least comparable to those that have previously been reported for the S. pyogenes CRISPR/Cas system. Stable inheritance of the induced targeted mutations of the ADH1 gene was demonstrated for both St1Cas9-and SaCas9-based systems at high frequencies. We were also able to demonstrate that the SaCas9 and SpCas9 proteins enhance homologous recombination via the induction of double-strand breaks only in the presence of their species-specific single guide (sg) RNAs. These proteins are not prone to inter-species interference with heterologous sgRNA expression constructs. Thus, the CRISPR/Cas systems of S. pyogenes and S. aureus should be appropriate for simultaneously addressing different sequence motifs with different enzyme activities in the same plant cell.

Rapid Evolution of Manifold CRISPR Systems for Plant Genome Editing

“…Recently, StCas9 and SaCas9 mediated genome editing was shown in Arabidopsis (Steinert et al, 2015), and SaCas9 mediated genome editing was reported in tobacco and rice (Kaya et al, 2016). These orthogonal Cas9 systems use different Cas9 proteins, PAM requirements and gRNA scaffolds for target recognition, hence expanding the targeting sites defined by the most popular SpCas9 system.…”

Applications and roles of the CRISPR system in genome editing of plants

“…Editing plant genomes without introducing foreign DNA into cells may alleviate regulatory concerns related to genetically modified plants. Transformed preassembled complexes of purified Cas9 protein and guide RNA into plant protoplasts of A. thaliana have achieved targeted mutagenesis in regenerated plants at frequencies of up to 46 % with the targeted sites contained germline-transmissible small insertions or deletions (Steinert et al 2015;Woo et al 2015).…”

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

“…However, SpCas9 variants were initially designed for applications in mammalian cells and have not yet been tested in plants [38]. Regarding SpCas9 orthologs, StCas9 and SaCas9 have recently been used to trigger mutagenesis in A. thaliana with high efficiency and without any cross interference between nucleases and sgRNA from different species [52]. SaCas9 which showed the highest mutation efficiency (90%) seems to be the most promising for CRISPR-induced gene knock-in.…”