Extended Data Fig. 8: Control reactions for integration assay and generation of 32P-labelled ladder.
a, Scheme of nine CRISPR fragments used for in vitro integration assays. Each CRISPR locus contains two repeats and two spacers. Leader motifs are color-coded and annotated (IR, inverted repeat; DR, direct repeat; IHF, IHF binding site; LAS, Leader anchoring site). To simplify the pictograms shown here and in subsequent panels, a single-colored rectangle was used to represent a given collection of leader motifs, and a single diamond was used to represent a CRISPR locus composed of two repeats and two spacers. b, The four CRISPR repeats tested in the integration assays have diverse palindromes (yellow), and a wide range of GC-content. c, Control reactions in which all components necessary for integration, except Cas1-2/3, were incubated. The overexposed gels show that the majority of the 32P signal for a given integration substrate DNA corresponds to the full-length strands. d, A custom 32P-labelled DNA ladder was made by mixing the degradation products generated by individual restriction enzyme digests of different 32P-labelled integration substrate DNAs. X1 and X2 signify integration substrates that were not analyzed for this manuscript. e, Schematics of the nine CRISPRs tested in Extended Data Fig. 5. The arrows identify locations of off-target integration reactions. Most off-target integration reactions occur by spurious integration at DNA motifs (for example, second CRISPR repeat, IHFdistal site, or upstream motifs) found near the ends of the CRISPR DNA target. Previous deep-sequencing of similar integration reactions has shown that the second repeat is a common off-target integration site, and that IHF blocks integration at the IHF binding site22. Urea-PAGE gels were run once.
