Publication:
Distance-dependent effects on CRISPR/Cas9-mediated genome editing in Schizosaccharomyces pombe compromise efficiency and create unsought alleles.
Authors
Protacio RU, Malone EG, Wahls WP
Community curation provided by Emory Malone
Added to PomBase on 2024-10-02
Citation
MicroPubl Biol 2024;2024
ID
PMID:39132053
Links
Abstract
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.
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