Implementation of the CRISPR-Cas9 system in fission yeast

Abstract

Application of the CRISPR-Cas9 genome editing system in the model organism Schizosaccharomyces pombe has been hampered by the lack of constructs to express RNA of arbitrary sequence. Here we present expression constructs that use the promoter/leader RNA of K RNA (rrk1) and a ribozyme to produce the targeting guide RNA. Together with constitutive expression of Cas9, this system achieves selection-free specific mutagenesis with efficiencies approaching 100%. The rrk1 CRISPR-Cas9 method enables rapid and efficient genome manipulation and unlocks the CRISPR toolset for use in fission yeast.

Figure 1

sgRNA expression system. (a) Schema of sgRNA expression construct and processing. HHR: Hammerhead Ribozyme. (b) 5′ RACE sequence. Only 4bp of the oligodG attached to the 5′ end, resulting from the method, are shown. (c) Northern analysis of Cas9 expressing , ade6+ strains with either no sgRNA vector or a sgRNA vector targeted against ade6-M210. Marker sizes are shown to the left and hybridization probe below. (d) ade6 targeting sequences.

Figure 2

ade6 mutagenesis. The original genotype and phenotype are shown in the first column, the sgRNA expression vector in the second, the PCR product used as HR template in the third, followed by average mutation efficiency, standard deviation and number of experiments. The phenotype of the survivor colonies is shown in stacked bar format, with error bars depicting standard deviation.