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

Sito Torres-Garcia,Lorenza Di Pompeo,Baptiste Gaborieau,Imtiyaz Yaseen,Alison L Pidoux,Sharon A White,Luke Eivers,Robin C Allshire,Yizhi Cai,Paulina Kanigowska

doi:10.12688/wellcomeopenres.16405.1

Sito Torres-Garcia, Lorenza Di Pompeo + Show 8 more

Open Access

https://doi.org/10.12688/wellcomeopenres.16405.1

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Abstract

The CRISPR/Cas9 system allows scarless, marker-free genome editing. Current CRISPR/Cas9 systems for the fission yeast Schizosaccharomyces pomberely 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 adh1promoter. To overcome these problems we have developed an improved system, SpEDIT, thatuses a synthesised Cas9 sequencecodon-optimised for S. pombeexpressed from the medium strength adh15promoter.The SpEDITsystem 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 SpEDITsystem allowed a 100% mutagenesis efficiency to be achieved when generating targeted pointmutants in the ade6 + or ura4 +genes by transformation of cells from asynchronous cultures. SpEDITalso 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 SpEDITsystem displayed reduced toxicity compared to currently available S. pombeediting systems. Thus, SpEDITprovides an effective and user-friendly CRISPR/Cas9 procedure that significantly improves the genome editing toolbox for fission yeast.

Highlights

The fission yeast Schizosaccharomyces pombe is a powerful model organism widely used in cellular and molecular biology (Fantes & Hoffman, 2016)
To circumvent issues pertaining to Cas9 toxicity and inefficient single-guide RNA (sgRNA) cloning procedures, here we report the development of SpEDIT, an improved CRISPR/Cas9 system for the efficient manipulation of the fission yeast genome
The SpEDIT system The SpEDIT system has been developed to address the two main complications associated with existing CRISPR/Cas9 methods for S. pombe: toxicity associated with Cas9 overexpression, and laborious cloning procedures required to insert a specific sgRNA target sequence into a Cas9-containing plasmid

Summary

Introduction

The fission yeast Schizosaccharomyces pombe is a powerful model organism widely used in cellular and molecular biology (Fantes & Hoffman, 2016). DNA constructs vary depending on the application but commonly consist of a sole PCR product that comprises an insertion, deletion or tagging cassette amplified from an existing plasmid (Bähler et al, 1998). Albeit convenient, this approach results in a selectable marker integrated at the target locus, disrupting the local genomic context and limiting the availability of markers for subsequent manipulations. The prokaryotic CRISPR/Cas system enables flexible and scarless genome editing without the necessity of selectable markers escorting the introduced DNA change and disturbing the local genomic environment (Hsu et al, 2014; Jinek et al, 2012). HDR involves recombination via sequence homology and can be exploited to generate precise mutations by providing a DNA editing template that contains the required DNA change and engages in homologous recombination (HR) with the cleaved region (Hsu et al, 2014)

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