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Highly efficient heritable plant genome engineering using Cas9 orthologues from Streptococcus thermophilus and Staphylococcus aureus
Author(s) -
Steinert Jeannette,
Schiml Simon,
Fauser Friedrich,
Puchta Holger
Publication year - 2015
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.13078
Subject(s) - cas9 , biology , crispr , genome , streptococcus pyogenes , genetics , genome editing , streptococcus thermophilus , mutagenesis , gene , genome engineering , homologous recombination , guide rna , computational biology , staphylococcus aureus , mutation , bacteria , lactobacillus
Summary The 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 ADH 1 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) RNA s. These proteins are not prone to inter‐species interference with heterologous sg RNA 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.