Extended Data Fig. 9: Additional analyses relating to RNA–seq data.

a, RNA enrichment measured by reverse transcription (RT)–qPCR of target-E RNA–seq sample (Fig. 5b) for two distinct primer pairs (left). The targeting condition (T) reveals around 140-fold enrichment of transcripts versus non-targeting (NT) condition. Standard curves for simulated gene activation to determine the limit of detection for primer pair 1 (center) and primer pair 2 (right) suggest a detection limit of around 5%. b, RNA–seq coverage plots for the target-E off-target site near tyrS, shown in Fig. 5f. The approximately 46 bp distance between TAM and TSS upstream of tyrS is indicated. Zoom-out view (right) shows pdxY is upregulated due to it being encoded directly downstream of tyrS. The TAM and extensive 11-bp complementarity between the off-target DNA site and guide sequence are shown below the coverage track. Coverage is shown for the reverse strand as counts per million (CPM). c, TSS plots derived from RNA-seq data, as shown in Fig. 5c, for all the individual gRNAs in Fig. 5g. Coverage is shown as CPM. d, RNA–seq tracks for a NT control and three distinct gRNAs designed for target-3 showing weak or no transcription initiation, likely due to binding site occlusion by other DNA binding factors involved in yidX regulation. Coverage is shown for the forward strand as CPM. NT, non-targeting. e, RNA–seq coverage plots for a gRNA designed for Target 6 (as shown in Fig. 5g) and three additional gRNAs that incrementally shift the TAM by 1 bp, changing it to A, T, and C. Targets flanked by a non-G-TAM fail to initiate transcription. Coverage is shown for the forward strand as CPM.