SpEDIT: A fast and efficient CRISPR/Cas9 method for fission yeast
Sito Torres-Garcia, Lorenza Di Pompeo, Luke Eivers, Baptiste Gaborieau, Sharon A. White, Alison L. Pidoux, Paulina Kanigowska, Imtiyaz Yaseen, Yizhi Cai, Robin C. Allshire
Abstract
<ns3:p> The CRISPR/Cas9 system allows scarless, marker-free genome editing. Current CRISPR/Cas9 systems for the fission yeast <ns3:italic>Schizosaccharomyces pombe </ns3:italic> 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 <ns3:italic>adh1 </ns3:italic> promoter. To overcome these problems we have developed an improved system, <ns3:italic>SpEDIT</ns3:italic> , that uses a synthesised Cas9 sequence codon-optimised for <ns3:italic>S. pombe </ns3:italic> expressed from the medium strength <ns3:italic>adh15 </ns3:italic> promoter. The <ns3:italic>SpEDIT</ns3:italic> 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 <ns3:italic>Bsa</ns3:italic> I type IIS restriction enzyme flanking a GFP placeholder enables one-step Golden Gate mediated replacement of GFP with synthesized sgRNAs for expression. The <ns3:italic>SpEDIT</ns3:italic> system allowed a 100% mutagenesis efficiency to be achieved when generating targeted point mutants in the <ns3:italic> ade6 <ns3:sup>+</ns3:sup> </ns3:italic> or <ns3:italic>ura4</ns3:italic> <ns3:sup>+</ns3:sup> genes by transformation of cells from asynchronous cultures. <ns3:italic>SpEDIT</ns3:italic> 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 <ns3:italic>SpEDIT</ns3:italic> system displayed reduced toxicity compared to currently available <ns3:italic>S. pombe</ns3:italic> editing systems. Thus, <ns3:italic>SpEDIT </ns3:italic> provides an effective and user-friendly CRISPR/Cas9 procedure that significantly improves the genome editing toolbox for fission yeast. </ns3:p>