Unexpected insertion of carrier DNA sequences into the fission yeast genome during CRISPR–Cas9 mediated gene deletion

Abstract

Abstract Objectives The fission yeast Schizosaccharomyces pombe is predicted to encode ~ 200 proteins of < 100 amino acids, including a number of previously uncharacterised proteins that are found conserved in related Schizosaccharomyces species only. To begin an investigation of the function of four of these so-called microproteins (designated Smp1–Smp4), CRISPR–Cas9 genome editing technology was used to delete the corresponding genes in haploid fission yeast cells. Results None of the four microprotein-encoding genes was essential for viability, meiosis or sporulation, and the deletion cells were no more sensitive to a range of cell stressors than wild-type, leaving the function of the proteins unresolved. During CRISPR–Cas9 editing however, a number of strains were isolated in which additional sequences were inserted into the target loci at the Cas9 cut site. Sequencing of the inserts revealed these to be derived from the chum salmon Oncorhynchus keta, the source of the carrier DNA used in the S. pombe transformation.

Keywords

• Microprotein
• Fission yeast
• Schizosaccharomyces pombe
• Oncorhynchus keta
• CRISPR–Cas9