Distance-dependent effects on CRISPR/Cas9-mediated genome editing in  Schizosaccharomyces pombe  compromise efficiency and create unsought alleles.
Discrete DNA sites position meiotic recombination at hotspots. We sought to create four different, 15 bp long, candidate regulatory DNA sites within the  ura4  reporter gene. Each effort employed a fission yeast-optimized CRISPR system (SpEDIT), optimal guide RNA, and one of four homologous recombination templates with 10 to 15 bp substitutions. Remarkably, every Ura  -  transformant analyzed had template-directed, PAM-disabling bp substitutions near (5-6 bp away from) the DSB but no DNA site-generating substitutions at distance (42-56 bp). An unsought novel allele,  ura4-P127*  , has two substitutions (C379T, C380A) that create a stop codon, rendering strains unable to grow without uracil.
Protacio RU, Malone EG, Wahls WP