Ciccaglione, Keith M.. Adaptation of CRISPR/Cas9 to improve the experimental utility of the model system schizosaccharomyces pombe. Retrieved from https://doi.org/doi:10.7282/T3N58P92
DescriptionSchizosaccharomyces pombe is a model organism that is utilized in several areas of research including DNA and chromatin biology. However, methods for genomic manipulation of the organism are outdated and cumbersome. The CRISPR/Cas9 toolset was recently developed from the prokaryotic type II CRISPR-associated system (a form of adaptive immunity) and has been shown to be successful at site-specific mutagenesis and modulation of gene expression in multiple eukaryotic organisms. We sought to modify the two components of this instrument, Cas9 and short-guide RNA (sgRNA), for effective use in S. pombe. As proof of concept, the ade6 gene was targeted for CRISPR/Cas9 mutagenesis by employing two constructs: one with an adh1 expressed Cas9 and one with sgRNA expressed by rrk1, the promoter for K RNA, and precisely terminated by a Hammerhead ribozyme. The ade6 gene, a long terminal repeat (LTR) sequence of the retrotransposons Tf1 and Tf2, and TER1 were also targeted by the rrk1 sgRNA and the catalytically null Cas9 (dCas) for site-specific enrichment of DNA sequence through chromatin immunoprecipitation. We found some Cas9 expressing vectors were inactivated by random mutation and studied this phenomenon using the inducible nmt1 promoter (no mRNA transcription in thiamine). Cultures with either nmt1:Cas9 or nmt1:dCas9 repressed by thiamine grew 4.4 to 7 times (p-value= 2.1x10-15 and 2.2x10-16, respectively) faster than those with full-expression. Toxicity was not attributable to the catalytic activity of Cas9 and further investigation is required. Cas9 and sgRNA were combined into a single expression vector to reduce exposure to inactivating mutations and yielded mutagenesis efficiencies of 86-92%. Also, by reducing expression of nmt1:dCas9 in thiamine, we achieved 50% to 300% enrichment of genomic target sites. The described rrk1 expressed sgRNA, with Cas9 and dCas9, will enable more efficient genomic manipulation of S. pombe.