a, Top, genomic locus targeted by crRNA-3 and crRNA-4, including both potential transposition products and the PCR primer pairs to selectively amplify them. Bottom, NGS analysis of the distance between the Cascade target site and transposon insertion site for crRNA-3 (left) and crRNA-4 (right), determined with two alternative primer pairs. b, Top, schematic of the lacZ locus with or without integrated transposon after transposition experiments with crRNA-4. T-LR and T-RL denote transposition products in which the transposon left end and right end are proximal to the target site, respectively. Primer pairs g and h (external–internal) selectively amplify the integrated locus, whereas primer pair i (external–external) amplifies both unintegrated and integrated loci. Bottom, PCR analysis of 10 colonies after 24-h growth on +IPTG plates (left) indicates that all colonies contain integration events in both orientations (primer pairs g and h), but with efficiencies sufficiently low that the unintegrated product predominates after amplification with primer pair i. After resuspending cells, allowing for an additional 18 h of clonal growth on −IPTG plates, and performing the same PCR analysis on 10 colonies (right), 3 out of 10 colonies now exhibit clonal integration in the T-LR orientation (compare primer pairs h and i). The remaining colonies show low-level integration in both orientations, which presumably occurred during the additional 18-h growth owing to leaky expression. These analyses indicate that colonies are genetically heterogeneous after growth on +IPTG plates, and that RNA-guided DNA integration only occurs in a proportion of cells within growing colonies. I, integrated product; U, unintegrated product. Asterisk denotes mispriming product also present in the negative (unintegrated) control. c, Photograph of LB-agar plate used for blue–white colony screening. Cells from IPTG-containing plates were replated on X-gal-containing plates, and white colonies expected to contain lacZ-inactivating transposon insertions were selected for further characterization. d, PCR analysis of E. coli strains identified by blue–white colony screening that contain clonally integrated transposons, as in b. e, Schematic of Sanger sequencing coverage across the lacZ locus for strains shown in d. f, PCR analysis of transposition experiment with crRNA-4 after serially diluting lysate from a clonally integrated strain with lysate from a control strain to simulate variable integration efficiencies, as in b. These experiments demonstrate that transposition products can be reliably detected by PCR with an external–internal primer pair at efficiencies above 0.5%, but that PCR bias leads to preferential amplification of the unintegrated product using the external-external primer pair at any efficiency substantially below 100%. For gel source data, see Supplementary Fig. 1.