MATERIALS AND METHODSMedia Sch. pombe strains were cultured in rich YES medium or chemically defined PMG medium supplied with the necessary supplements. PMG5 medium contains all five supplements, adenine, histidine, leucine, uracil, and lysine, at 225 mg/l, and drop-out media were prepared by leaving out one of the five supplements. In the PMG5 with low adenine medium, the adenine concentration was reduced to 22.5 mg/l. Top10 E. coli were grown in Luria Broth medium. In order to select bacteria with drug-resistant genes, carbenicillin (Sigma-Aldrich) or kanamycin (Sigma-Aldrich) were used at a final concentration of 75 or 50 mg/ml, respectively. Agar was added to 2% for preparation of solid media.PlasmidsAll plasmids constructed in this study are listed in Supplementary Table S1, including their Addgene accession number if applicable. The original plasmid with the FnCas12a codon optimized for human was obtained from Addgene (#103008) (40). Then, the FnCas12a CDS with nucleoplasmin nuclear localization signal (NLS) was assembled with adh1 promoter and CYC1 terminator by Golden Gate cloning. The whole cassette of Padh1-FnCas12a-TCYC1 was inserted into the empty vector with ura4 marker and ars1 replicating region. To build the initial crRNA expression module, the rrk1 promoter with direct repeat, NotI cutting site and hammerhead ribozyme (HHRz) were synthesized as a gBlock from IDT. To build the crRNA expression module with more pol II promoters from endogenous protein coding genes, we PCR amplified the promoter regions (around 1kb upstream from the start codon) and assembled them with direct repeats, a BsaI entry pad and TEF1 terminators using Golden Gate cloning. The resulting crRNA expression module was inserted into the plasmids with the FnCas12a cassette, to serve as the final entry vector.To build the functional plasmid for genomic editing, a single gRNA or one gRNA array was inserted into the entry vector following similar strategies with previous reports (10). All gRNAs were designed using online tool CRISPOR (41). For the single gRNA, two complementary primers with gRNA sequence and BsaI adapter were ordered from IDT, annealed together, and then ligated to the entry vector that had been pre-digested with BsaI enzyme. For multiplex genomic editing, the gRNA array was ordered from IDT as a gBlock or multiple primers for annealing, then assembled into entry vectors with Golden Gate clone. A detailed protocol for gRNA clone is included as Supplementary Figure S1. All gRNA sequences used in this study are listed in Supplementary Table S2. The primers used to assemble the gRNAs into entry vectors are listed in Supplementary Table S3. The crRNA expression module sequence (Pfba1-crRNAarray-TTEF1) is in Supplementary Table S4.For the plasmid with dCas12a, the D917A mutation was introduced into FnCas12a but the other components remain identical (8,42). To build the dCas12a fused with Clr4 DNA methyltransferase, the Clr4 enzymatic domain was PCR amplified from wild type Sch. pombe genomic DNA and assembled into the C-terminus of dCas12a. An additional SV40 NLS was fused to the N-terminus. To build the dCas12a fused with catalytic dead Clr4 domain, we used a two-step fusion PCR to introduce point mutations (G378S or G486D) into the Clr4 catalytic domain of dCas12a-Clr4 construct.Yeast transformation and genomic editingAll fission yeast transformations were performed following a LiOAc transformation protocol (30). BP232 (h– ura4D-18) was used as the parent strain to test all the Cas12a genome editing efficiency. The cells were grown in PMG5 medium to an A600 between 0.45 and 0.5, and then harvested by centrifugation. The transformation efficiency was around 104 CFU/μg DNA. For genomic DNA editing, 2 μg of FnCas12a/crRNA plasmids were transformed, together with 2 μg linearized plasmid or PCR products as donor DNA if necessary. To compare editing efficiency of different gRNA sequences or the same gRNA driven by different promoters, we performed each group's experiments with replicates in parallel by using the same parent strain, transforming the same amount of plasmid and donor DNA. For multiplex genomic editing experiments, 2 μg donor DNA was used for each. To ensure efficient homologous recombination, usually 800bp homologous region was used on each side. Detailed homology lengths tested in this study are listed in Supplementary Table S5. After transformation, cells were spread on PMG5–Ura plates, and incubated at 30°C for 6–7 days to form single colonies. To check editing efficiency at ade6, cells were spread on PMG5–Ura plates with low adenine concentration (22.5 mg/l). For other auxotrophic markers, the plates with single colonies were replica-plating to the corresponding drop out media to check auxotrophy. Genomic editing was further confirmed by colony PCR and Sanger sequencing.To build yeast strains with synonymous mutations in the ade6 CDS, we first cloned the WT ade6 CDS with homology arms into a Blunt II-Topo vector via Zero Blunt Topo Cloning (ThermoFisher #450245) and introduced these point mutations by fusion PCR. To build the corresponding yeast strains, we started with the ade6Δ0 strain obtained from our first CRISPR editing experiments that targeted ade6+. Then, we transformed the DNA with synonymous mutations in ade6 and homology arms into the ade6Δ0 strain and selected on PMG5–Ade plates. These ‘synonymous’ strains were used to testCas12a PAM preference.
Article TitleCRISPR–Cas12a system in fission yeast for multiplex genomic editing and CRISPR interference
All plasmids used in this study are listed inSupplementary Table S1and are available upon request. The entry vectors for CRISPR genome editing with Cas12a were deposited to Addgene as accession number #132952 for pYZ221 and #132953 for pYZ673. The entry vectors for CRISPRi with dCas12a have been deposited to Addgene as #132954, #132955, #132956 for pYZ675, pYZ714 and pYZ694 respectively.