Creating Meiotic Recombination-Regulating DNA Sites by SpEDIT in Fission Yeast Reveals Inefficiencies, Target-Site Duplications, and Ectopic Insertions

Recombination hotspot-activating DNA sites (e.g., <i>M26</i>, <i>CCAAT</i>, <i>Oligo-C</i>) and their binding proteins (e.g., Atf1-Pcr1 heterodimer; Php2-Php3-Php5 complex, Rst2, Prdm9) regulate the distribution of Spo11 (Rec12)-initiated meiotic recombination. We sought to create 14 different candidate regulatory DNA sites via bp substitutions in the <i>ade6</i> gene of <i>Schizosaccharomyces pombe</i>. We used a fission yeast-optimized CRISPR-Cas9 system (<i>SpEDIT</i>) and 196 bp-long dsDNA templates with centrally located bp substitutions designed to ablate the genomic PAM site, create specific 15 bp-long DNA sequences, and introduce a stop codon. After co-transformation with a plasmid that encoded both the guide RNA and Cas9 enzyme, about one-third of colonies had a phenotype diagnostic for DNA sequence changes at <i>ade6</i>. PCR diagnostics and DNA sequencing revealed a diverse collection of alterations at the target locus, including: (A) complete or (B) partial template-directed substitutions; (C) non-homologous end joinings; (D) duplications; (E) bp mutations, and (F) insertions of ectopic DNA. We concluded that <i>SpEDIT</i> can be used successfully to generate a diverse collection of DNA sequence elements within a reporter gene of interest. However, its utility is complicated by low efficiency, incomplete template-directed repair events, and undesired alterations to the target locus.