Fig. 2: A simple distance metric does not predict CRISPRa activity.

a CRISPRa on the aroK-aroB operon. Two scRNA target sites within the 40 base window where CRISPRa is effective (−100 to −60) in heterologous reporter genes (A3–A4) and two sites further upstream (A1–A2) were chosen for the aroKp1 promoter. b CRISPRa on the cysK gene. Three scRNA target sites within the 40 base window where CRISPRa is effective in heterologous reporter genes (C1–C3) and two sites further downstream (C4–C5) were chosen for the cysKp2 promoter. The C4 and C5 sites resulted in repression; targeting these sites close to the core promoter may interfere with RNA polymerase binding. Gene expression was measured using RT-qPCR. Fold activation represents expression levels relative to a strain expressing an off-target scRNA (hAAVS1). In a and b, bars represent the average ± standard deviation calculated from n = 4 (C1, C2), n = 3 (A1–4, C4,C5), or n = 2 (C3) biologically independent samples. Some individual replicates in samples A1–4 and C1–2 appear to show activation, but a two-tailed unpaired Welch’s t test indicates that the average differences relative to the off-target control are not statistically significant (p value > 0.05). Targeting CRISPRa to C4–5 resulted in repression, likely owing the short distance between the sites and the promoter. Stars indicate a statistically significant difference from the off-target control using a two-tailed unpaired Welch’s t test (**p value < 0.01). Exact p values: A1: 0.18, A2: 0.27, A3: 0.36, A4: 0.17, C1: 0.29, C2: 0.66, C3: 0.098, C4: 0.0022, C5: 0.00043. Source data are provided as a Source Data file.