All plasmid cleavage experiments were initiated by co-transforming electrocompetent US0 cells with a (d)Cas9 expression plasmid and a single-target plasmid. For clonal plasmid cleavage assays, 1 µl of each plasmid miniprep was co-delivered, and cells were recovered with rotation at 37 °C in 15 mL conical-bottom tubes containing 10 mL SOB supplemented with glucose (0.5%). After 1.25 h recovery, cells were serially diluted and plated on media with antibiotics to select for both plasmids (kanamycin and carbenicillin) or only the Cas9 plasmid (carbenicillin). Plates were supplemented with IPTG or glucose as indicated in the figures and photographed after 16 h at 37 °C. For library cleavage (PAM-depletion) assays, 2 µl of Cas9 (or dCas9 control) plasmid minipreps were co-delivered with 1 µl of the PAM library maxiprep. These conditions inherently favor the formation of double transformants that have received only a single PAM library plasmid, because our unconcentrated Cas9 plasmid preps are typically in molar excess of pSC101-based (low-copy) target plasmid preps. After 1.25 h recovery as described above, cells were pelleted by centrifugation at 1900 × g for 10 min, washed twice in 2 mL tubes using fresh broth, then resuspended in a final volume of 1 mL for storage at 4 °C overnight. An aliquot was withdrawn prior to refrigeration for serial dilution and plating on 2xYT media supplemented with glucose (2%) and both antibiotics to quantify the total number of double transformants. Based on CarbRKanR CFU counts recorded the next day, 1.0e7 double transformants from each sample were spread on individual BioAssay plates containing 2xYT media supplemented with IPTG (10 µM) and both antibiotics. Plates were incubated for 10 h at 37 °C and then scraped for miniprepping. To introduce indices and Illumina adapters for multiplexed sequencing on a NextSeq 500, the target plasmid’s randomized regions were PCR-amplified in a single step (15 cycles) from the undiluted miniprep pools or pre-depletion PAM library maxiprep, using unique combinations of primers listed in Supplementary Data 5.

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