Extended Data Fig. 1 |. Design of Flag-tagged transpososome components and analysis of ChIP data.
a, Schematic highlighting the 3×Flag-tag positions in each protein component that was used for ChIP–seq experiments. Molecular weights are indicated in parentheses. b, Simplified schematic of the in vivo transposition assay used to quantify RNA-guided DNA integration in E. coli. Mini-transposon (Mini-Tn) DNA substrates are mobilized from pDonor to genomic DNA, downstream of the target site complementary to crRNA, and integration is quantified by junction PCR. c, RNA-guided transposition assays for 3×Flag-tagged constructs demonstrate that near-wild-type activity is maintained. A single point mutation in the TnsB active site (D223N) completely ablates DNA integration. TnsA-Flag was not pursued further after weak enrichment at the target site was observed (see panel d), and hence was only tested once. NT, non-targeting crRNA control. d, ChIP–seq data for 3×Flag-tagged TnsA with crRNA-4, shown as in Fig. 1c, d, reveal inefficient DNA enrichment at the genomic target site (left). However, the prominent peaks at both transposon ends on pDonor (right) indicate that the protein retains in vivo function and likely exhibits inefficient crosslinking to the target DNA within the pre-integration transpososome complex. Read coverage (RPKM) is scaled to the highest peak (E. coli genome mapping reads) or TnsB ChIP-seq data (pDonor mapping reads). e, Representative ChIP–seq data for experiments with crRNA-287, shown as in Fig. 1c, d, reveal accurate recruitment of multiple distinct factors to the corresponding genomic target site. f, ChIP–seq data reveal robust self-targeting of the CRISPR array across multiple crRNA datasets. Reads mapping to pQCascade are shown for two distinct crRNAs for the indicated Flag-tagged components, and were normalized to the highest peak, except for input samples, which were normalized to the highest peak in their corresponding Cas8 sample. The vector map of pQCascade is shown (top), and the position of the CRISPR spacer is denoted with maroon triangles (bottom). Non-unique reads mapping to lacI were not analysed (dotted lines). g, CRISPR interference (CRISPRi)-based repression of genomically-encoded mRFP using Cascade in the presence (light blue) or absence (ΔTniQ, dark blue) of TniQ. Transcriptional repression is measured as OD-normalized mRFP fluorescence relative to the perfectly matching (PM) crRNA. NT, non-targeting; 1–4, crRNA mismatches at position 1–4; 25–28, crRNA mismatches at position 25–28. h, Target DNA binding by Cascade (crRNA-4) with and without TniQ, as measured by fluorescence polarization. Colouring is as shown in g. i, TnsC may be stabilized in the pre-integration transpososome complex by the presence of TnsAB. ChIP–seq reads mapping to a 3-kb window centred at the genomic target site are shown for 3×Flag-tagged TnsC in experiments that differed only in the presence or absence of TnsA and TnsB (top), with read coverage (RPKM) scaled the same in both graphs. Note that the same data are presented in Fig. 1f. Quantitative PCR was performed on the same ChIP samples alongside a non-targeting control, confirming the diminished target DNA enrichment levels when TnsA-TnsB were omitted (bottom). Relative enrichment is calculated as ΔΔCq between the target locus and reference gene. j, TnsB can be recruited to the RNA-guided target site in the absence of TnsA and donor DNA, albeit with reduced efficiency. ChIP–seq reads mapping to a 3-kb window centred at the genomic target site are shown for 3×Flag-tagged TnsB in experiments that differed only in the presence or absence of TnsA and pDonor, with read coverage (RPKM) scaled the same in all three graphs. Data in c, g, h and i are shown as mean ± s.d. of n = 3 independent biological replicates, except for TnsA-Flag (c, n = 1) and ΔTnsAB (i, n = 1).