Fig. 3

Schematic representation of the pFC332_Pro¹-sgRNA plasmid construction. a Amplification of the two flanks that represent the Pro¹-sgRNA expression cassette: pTE1_for and pRC-target are used to amplify the Pro¹-tRNA promoter and target sequence, where pRC-target contains a variable 20 bp overhang (indicated by brown color) that represents the reverse complement target sequence of choice. In turn, pTarget and pTE1_rev are used to amplify the target-sgRNA-Pro¹-tRNA terminator flank. Here, pTarget contains a variable overhang that contains the target sequence of choice. Separate flanks are joined together through fusion PCR by pTE1_for and pTE1_rev, where the overhang sequence (=target) facilitates the homologous region between both flanks. b Addition of PacI sites to either end of the fusion construct (part of pTE1_for and pTE1_rev sequence) allows ligation of the fusion construct into pFC332. Diagnostic restriction analysis of the cloned plasmid ought to be done with Cfr42i (SacII) and shows a fragment of either 497 bp or 500 bp in addition to 1 kb and 14.3 kb fragments, for forward or reverse orientation, respectively