A CRISPR/Cas9-based method and primer design tool for seamless genome editing in fission yeast

Abstract

In the fission yeast Schizosaccharomyces pombe the prevailing approach for gene manipulations is based on homologous recombination of a PCR product that contains genomic target sequences and a selectable marker. The CRISPR/Cas9 system has recently been implemented in fission yeast, which allows for seamless genome editing without integration of a selection marker or leaving any other genomic ‘scars’. The published method involves manual design of the single guide RNA (sgRNA), and digestion of a large plasmid with a problematic restriction enzyme to clone the sgRNA. To increase the efficiency of this approach, we have established and optimized a PCR-based system to clone the sgRNA without restriction enzymes into a plasmid with a dominant natMX6 (nourseothricin) selection marker. We also provide a web-tool, CRISPR4P, to support the design of the sgRNAs and the primers required for the entire process of seamless DNA deletion. Moreover, we report the preparation of G1-synchronized and cryopreserved S. pombe cells, which greatly increases the efficiency and speed for transformations, and may also facilitate standard gene manipulations. Applying this optimized CRISPR/Cas9-based approach, we have successfully deleted over 80 different non-coding RNA genes, which are generally lowly expressed, and have inserted 7 point mutations in 4 different genomic regions.

Construction of Designer Selectable Marker Deletions with a CRISPR-Cas9 Toolbox in Schizosaccharomyces pombe and New Design of Common Entry Vectors

“…Deletion efficiency was 10–20% and all positive colonies were relatively small in size. This was consistent with a previous report of Cas9 toxicity in fission yeast ( Rodríguez-López et al 2017 ). We hypothesized that slower growth was caused by Cas9 protein toxicity, as bigger colonies turned out to be negative for the deletion, possibly because Cas9 was inactive.…”

Atypical meiosis can be adaptive in outcrossedS. pombedue towtfmeiotic drivers

“…To delete Sp wtf4, we utilized the CRISPR/Cas9-based method described in Rodriguez-Lopez et al, 2016. We first cloned a plasmid (pSZB570) encoding Cas9 and a guide RNA targeting Sp wtf4 .…”

Uncovering Natural Longevity Alleles from Intercrossed Pools of Aging Fission Yeast Cells

“…We first used a more distal PAM to precisely target the region containing the indel for deletion, and then targeted the deleted region for reinsertion of a template containing the alternative allele (Figure S5). Deletions of ∼200 bp were made as previously described ( Rodríguez-López et al 2017 ), with the small addition of an arbitrary modification in the HR template (Figure S5, A–C). The modification was designed to neighbor a PAM site lying at the edge of the deletion.…”