Plasmid Creation via CPEC Cloning

All plasmids listed in Table S1 were created using circular polymerase extension cloning (CPEC)24 (link). The primers listed in Table S2 were used to create linear dsDNA products that were subsequently DpnI digested for at least 15 minutes and then gel-purified. The DNA was eluted in 6 ul of dH₂O and mixed with corresponding linear product. These products were then used as template for a reaction with Q5 polymerase for 15 cycles. The product was then used to directly transform chemically competent E. coli DH5α or NEB Turbo cells. The following plasmids and maps have been deposited to Addgene (Cambridge, MA); pCas9cr4 (Plasmid #62655), pKDsg-ack (Plasmid #62654), and pKDsg-p15 (Plasmid #62656).
The 20 bp targeting sequences of the sgRNA were re-targeted by CPEC cloning of two linear PCR fragments. The re-targeting primers listed in Table S2 were approximately 40-mers that had overlapping protospacer sequences. The primer pair protospacerF and gamR were used to yield a 3 kb product. The protospacer R primer was paired with pKDseq1F to yield a product of about 4 kb. This design yielded PCR product with about 280 bp of overlapping homology between the gam and araC (Fig. 2), as well as 20 bp of overlap in the protospacer. PCR products were gel purified, mixed together in equal volumes and CPEC cloned with Q5 polymerase. The mixture was used to transform chemically competent DH5α or NEB Turbo cells (New England Biolabs), recovered for 1 hour in super optimal broth with catabolite repression (SOC), and then plated on LB with 50 mg L⁻¹ spectinomycin (spec) and incubated at 30 °C. A step-by-step protocol for primer design and retargeting is available as part of the no-SCAR protocol at Addgene.