Construction of Deletion Mutants of DNA Polymerase Genes in S. islandicus

The CRISPR-based genetic manipulation method developed in our laboratory (Li et al., 2016) was employed to construct deletion mutants of S. islandicus for all four DNA polymerase genes (dpo), that is, dpo1, dpo2, dpo3, and dpo4. Genome-editing plasmids carrying a mini-CRISPR array with a spacer targeting each DNA polymerase gene and a donor DNA flanking the corresponding mutated allele were constructed as follows: (a) spacer DNA fragments were prepared by annealing of two complementary oligos (e.g., KOdpo2spf and KOdpo2spr). (b) Spacer fragments were individually inserted into the pSe-Rp vector [a vector carrying repeat sequences of CRISPR system (Peng et al., 2015) at the SapI site, giving pAC-dpo1, pAC-dpo2, pAC-dpo3, and pAC-dpo4 vectors]. (c) Donor DNAs were obtained by the splicing by overlap extension (SOE) PCR (Feng et al., 2018) in which the first set of primers (e.g., KOdpo2Larm-F and KOdpo2SOE-R) were used to yield the upstream part of the donor DNA, whereas the second set of primers (e.g., KOdpo2SOE-F and KOdpo2Rarm-R) were used to amplify the downstream part. These two DNA fragments were then spliced together by PCR using the primer combination of the forward primer of upstream fragment and the reverse primer of the downstream fragment, for example, KOdpo2Larm-F and KOdpo2Rarm-R primers, and this yielded donor DNAs carrying a deletion allele of each DNA polymerase gene. (d) Each donor DNA was inserted into the corresponding pAC-dpo vectors at the SphI and XholI sites, yielding genome-editing plasmid, pGE-dpo1, pGE-dpo2, pGE-dpo3, and pGE-dpo4, individually. The plasmids and DNA oligonucleotides employed in this work are listed in Supplementary Table S1 and S2.

Next, each genome-editing plasmid was introduced into S. islandicus E233S by electroporation, giving transformants on selective plates. Deletion mutant strains were identified by PCR amplification of each mutant allele using, for example, KOdpo2 F/KOdpo2 R and dpo2inner F/dpo2inner R primers and verified by DNA sequencing of deletant alleles. Finally, genome editing plasmids were cured from the mutants by counterselection for pyrEF marker using uracil and 5-FOA, yielding Δdpo2, Δdpo3, and Δdpo4 for further experiments.