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

Abstract

In the fission yeast the prevailing approach for Schizosaccharomyces pombe 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 (nourseothricin) selection marker. We also provide a web-tool, natMX6 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 cells, which S. pombe 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.

Pyruvate kinase variant of fission yeast tunes carbon metabolism, cell regulation, growth and stress resistance

“…We set out to analyse the effect of the pyk1 SNP on cellular metabolism. Using seamless CRISPR/Cas9‐based gene editing (Rodríguez‐López et al , ), we replaced the pyk1 T‐allele in the laboratory strain for the more common A‐allele. We introduced the A‐allele in both a heterothallic h − and a homothallic h 90 laboratory strain, without any other genetic perturbations.…”

“…The allele replacement strains were generated using the CRISPR‐Cas9 system. The plasmid containing the gRNA targeting the pyk1 SNP in 968 h 90 (homothallic) and 972 h − (heterothallic) was generated as described (Rodríguez‐López et al , ) using the following primers: gRNA.JB50‐F: GCTTTCCGGTGAGACTACCAgttttagagctagaaatagc and gRNA.JB50‐R: TGGTAGTCTCACCGGAAAGCttcttcggtacaggttatgt.…”

A cloning-free method for CRISPR/Cas9-mediated genome editing in fission yeast

“…As has been observed by others (Jacobs et al 2014;Rodriguez-Lopez et al 2016), the rare fast-growing transformant colonies, which appeared to have escaped from the growth inhibitory effect of Cas9, usually do not harbor the intended genome-editing outcome, probably because Cas9 is absent or not functional in these clones. Such colonies were avoided when picking random colonies for phenotype assessment (e.g.…”

“…In the published procedures of applying the CRISPR/Cas9 technology in fission yeast, a plasmid-cloning step is always undertaken each time a new sgRNA is needed (Jacobs et al 2014;Rodriguez-Lopez et al 2016;Fernandez and Berro 2016). This step is a multi-day endeavor that includes manipulations of DNA, transforming E. coli cells, plasmid preparation, and plasmid verification by sequencing.…”

A natural variant of the sole pyruvate kinase of fission yeast lowers glycolytic flux triggering increased respiration and oxidative-stress resistance but decreased growth

“…We set out to analyse the effect of the pyk1 SNP on cellular metabolism. Using seamless CRISPR/Cas9-based gene editing 54 , we replaced the pyk1 T-allele in the laboratory strain for the more common A-allele. We introduced the A-allele in both a heterothallic hand a homothallic h 90 laboratory strain, without any other genetic perturbations.…”