a, Schema depicting experimental work-flow. Cells were transfected with plasmids encoding dCas9-P300 and sgRNA variants. RNA collected from cells was used to measure gene activation of IL1RN, sgRNA expression levels and spacer sequence identities. b, Schema depicting 5′ RACE applied specifically to sequence sgRNAs. Template switching of the reverse transcriptase ensures accurate profiling of the 5′ ends of sgRNA variants. c, Gene activation induced by each sgRNA variant. This experiment was performed as shown in Fig. 1, except that extraction of total RNA, rather than only mRNA, was performed. d, Replotting the mean of each group in c as a function of the predicted folding energy of each hp-sgRNA’s engineered secondary structure. e, Expression level of each sgRNA variant as measured by RT–qPCR. f, Replotting the mean of each hp-sgRNA’s activity in c against its mean expression level as shown in e. g, The distribution of spacer lengths in cells treated with various sgRNA variants, as determined by 5′ RACE followed by deep sequencing. The number next to the sgRNA alias indicates the number of nucleotides added to the 5′ end of the spacer (for example, hp17 and ns17 have 17 nucleotides (nt) added, and a total spacer length of 37 nt). The expected unprocessed length of an sgRNA variant is highlighted in an orange box. h, Percentage of processing to 20 nt for each sgRNA variant, as measured by RNA-seq. i, Replotting the mean of each hp-sgRNA’s activity in c against its mean degree of processing as shown in i. The mean is the measure of center and error bars represent s.e.m. for n = 3. The sequences of all sgRNAs are listed in Supplementary Table 1.