This protocol is extracted from research article:
Minimal PAM specificity of a highly similar SpCas9 ortholog
Sci Adv, Oct 24, 2018; DOI: 10.1126/sciadv.aau0766

Plasmids for the SpCas9 sgRNA and PAM-SCANR genetic circuit, as well as BW25113 ΔlacI cells, were provided by the Beisel Lab (North Carolina State University). Plasmid libraries containing the target sequence followed by either a fully randomized 8-bp 5′-NNNNNNNN-3′ library or fixed PAM sequences were constructed by conducting site-directed mutagenesis on the PAM-SCANR plasmid flanking the protospacer sequence. Nuclease-deficient mutations (D10A and H850A) were introduced to the ScCas9 variants using Gibson Assembly. The provided BW25113 cells were made electrocompetent using standard glycerol wash and resuspension protocols. The PAM library and sgRNA plasmids, with resistance to kanamycin (Kan) and carbenicillin (Crb), respectively, were coelectroporated into the electrocompetent cells at 2.4 kV, outgrown, and recovered in Kan + Crb LB medium overnight. The outgrowth was diluted 1:100, grown to ABS600 of 0.6 in Kan + Crb LB liquid medium, and made electrocompetent. Indicated Cas9 plasmids, with resistance to chloramphenicol (Chl), were electroporated in duplicates into the electrocompetent cells harboring both the dCas9 and sgRNA plasmids, outgrown, and collected in 5 ml of Kan + Crb + Chl LB medium. Overnight cultures were diluted to an ABS600 of 0.01 and cultured to an OD600 (optical density at 600 nm) of 0.2. Cultures were analyzed and sorted on a FACSAria machine (Becton Dickinson). Events were gated on the basis of forward scatter and side scatter, and fluorescence was measured in the fluorescein isothiocyanate (FITC) channel (488-nm laser excitation, 530/30 filter for detection), with at least 30,000 gated events for data analysis. Sorted GFP-positive cells were grown to sufficient density, and plasmids from the presorted and sorted populations were then isolated, and the region flanking the nucleotide library was PCR-amplified and submitted for Sanger sequencing (Genewiz). Bacteria harboring nonlibrary PAM plasmids, performed in duplicates, were analyzed by FACS following electroporation and overnight incubation and represented as the percentage of GFP-positive cells in the population using SD to calculate error bars. The PAM-SCANR spacer sequence is 5′-CGAAAGGTTTTGCACTCGAC-3′. Additional details on the PAM-SCANR assay can be found in Leenay et al. (23).

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