 SpEDIT : A fast and efficient CRISPR/Cas9 method for fission yeast.
The CRISPR/Cas9 system allows scarless, marker-free genome editing. Current CRISPR/Cas9 systems for the fission yeast   Schizosaccharomyces pombe  rely on tedious and time-consuming cloning procedures to introduce a specific sgRNA target sequence into a Cas9-expressing plasmid. In addition, Cas9 endonuclease has been reported to be toxic to fission yeast when constitutively overexpressed from the strong   adh1  promoter. To overcome these problems we have developed an improved system,   SpEDIT , that uses a synthesised Cas9 sequence codon-optimised for   S. pombe  expressed from the medium strength   adh15  promoter. The   SpEDIT  system exhibits a flexible modular design where the sgRNA is fused to the 3' end of the self-cleaving hepatitis delta virus (HDV) ribozyme, allowing expression of the sgRNA cassette to be driven by RNA polymerase III from a tRNA gene sequence. Lastly, the inclusion of sites for the   Bsa I type IIS restriction enzyme flanking a GFP placeholder enables one-step Golden Gate mediated replacement of GFP with synthesized sgRNAs for expression. The   SpEDIT  system allowed a 100% mutagenesis efficiency to be achieved when generating targeted point mutants in the   ade6  +    or   ura4   +  genes by transformation of cells from asynchronous cultures.   SpEDIT  also permitted insertion, tagging and deletion events to be obtained with minimal effort. Simultaneous editing of two independent non-homologous loci was also readily achieved. Importantly the   SpEDIT  system displayed reduced toxicity compared to currently available   S. pombe  editing systems. Thus,   SpEDIT  provides an effective and user-friendly CRISPR/Cas9 procedure that significantly improves the genome editing toolbox for fission yeast.
Torres-Garcia S, Di Pompeo L, Eivers L, Gaborieau B, White SA, Pidoux AL, Kanigowska P, Yaseen I, Cai Y, Allshire RC