CRISPR/Cas9 editing plasmid construction.

Construction of a CRISPR/Cas9 genome editing plasmid was done in two cloning steps (Fig. S1). First, the spacer was incorporated into the CRISPR array via restriction digestion and ligation cloning. The plasmid was digested with BsaI-HF (NEB) in CutSmart buffer at 37°C. Meanwhile, the spacer was prepared to be ligated into the plasmid. First the oligonucleotides, ordered with the correct overhangs (57), were annealed by mixing the oligonucleotides at 10 μM each in 1× annealing buffer (10 mM Tris-Cl, pH 7.5, 100 mM NaCl, and 0.1 mM EDTA). Annealed oligonucleotides were phosphorylated using T₄ polynucleotide kinase (PNK; NEB). A 50-μl reaction mixture was assembled with 1× T₄ DNA ligase buffer, 10 U T₄ PNK, and 1 μM annealed oligonucleotides and incubated at 37°C for 30 min. T₄ PNK was heat inactivated at 65°C for 20 min. A 20-μl ligation reaction mixture was assembled using 1× T₄ DNA ligase buffer, 40 to 100 ng plasmid DNA, 25 nM phosphorylated and annealed oligonucleotides, and 400 units T₄ DNA ligase (NEB). The ligase reaction was performed at room temperature for 2 to 3 h. The ligation was then used to transform chemically competent (CaCl₂ method) E. coli cells (Top10; Thermo Fisher Scientific): 10 μl of the ligation reaction mixture was used to transform 60 to 100 μl competent cells. The plasmid was verified via Sanger sequencing. The resulting plasmid (essentially a targeting plasmid) was then used to create the editing plasmid.

The editing plasmid was constructed using Gibson Assembly (58) of the following four (or more) PCR amplicons that were gel purified from an agarose gel: (i) the vector backbone of pPB41, amplified using oPEB217/oPEB218; (ii) the Cas9/CRISPR array containing the spacer incorporated in the targeting plasmid, amplified using oPEB232/oPEB234; (iii) the upstream portion of the editing template, which contains overlaps for pPB41 at oPEB217 and for the downstream portion of the editing template (see pPB50 and pPB51 for examples); and (iv) the downstream portion of the editing template, which contains overlaps for the upstream portion of the editing template and for pPB41 at oPEB232 (see pPB50 and pPB51 for examples). A 10-μl Gibson Assembly reaction mixture was assembled using 1× Gibson Assembly master mix, 40 to 100 ng of the vector backbone of pPB41, 40 to 100 ng of the Cas9/CRISPR array containing the spacer, and about 20 to 40 ng of each portion of the editing template. The Gibson reaction was performed at 50°C for 90 min. The reaction was then used to transform chemically competent MC1061 E. coli cells. Clones were verified using Sanger sequencing at the University of Michigan Core Facility. The result of this second step yields a complete editing plasmid that can be used to efficiently manipulate the B. subtilis genome.