SpEDIT : A fast and efficient CRISPR/Cas9 method for fission yeast

Wellcome Open Research

article

Lorenza Di Pompeo¹  Luke Eivers¹  Baptiste Gaborieau¹  Sharon A. White¹  Alison L. Pidoux¹  Paulina Kanigowska²  Imtiyaz Yaseen¹  Yizhi Cai¹  Robin C. Allshire¹  Sito Torres-Garcia¹ 

[1] Wellcome Centre for Cell Biology and Institute of Cell Biology, School of Biological Sciences, University of Edinburgh;School of Biological Sciences, University of Edinburgh;Manchester Institute of Biotechnology, University of Manchester

关键词: CRISPR/Cas9;    gene editing;    gene tagging;    S. pombe;    fission yeast;   

DOI  :  10.12688/wellcomeopenres.16405.1

学科分类：内科医学

来源: Wellcome

【 摘 要 】

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 BsaI 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.

【 授权许可】

CC BY