Fig. 6: High-throughput guide selection through guide coupled barcode.

a Schematic of barcode approach. Briefly, a library of unique barcodes corresponding a specific spacer sequence was delivered to HEK293 cells with Rfx-dCas13d. Functional RNPs target to intron 10/11 of LMNA and undergo trans-splicing. RNA is harvested and the abundance of each barcode at the start of exon 11 was measured by targeted amplicon sequencing. This barcode corresponds to the guide associated with its trans-splicing. b enrichment plot for each guide targeting LMNA intron 10/11 as a function of position. Y-axis is barcode enrichment calculated by the (%barcode in trans-spliced RNA / %barcode in input plasmid DNA), and x-axis is the position along LMNA intron 10/11 of each guide. Each unique barcode (3) for a single guide is plotted as a triangle and the average of these enrichment scores is plotted as a black circle. The best guide is plotted in purple. c enrichment plot for each guide targeting DMPK intron 13/14 as a function of position. d Schematic of endogenous DMPK transcript (left) and edited DMPK transcript (right). Notably, there is a single G > T transition mutation installed between the edited transcript and the endogenous transcript. e Frequency of 3′CRAFT editing in endogenous DMPK transcripts. The y-axis is the percent of reads from targeted amplicon sequencing containing the snp mutation. The x-axis refers to specific treatment: dCas13 alone, rcRNA alone, dCas13 with an rcRNA that does not target the DMPK intron, and dCas13 with an rcRNA that targets the given intron (Data are mean ± s.d. from n = 3 individual samples). Statistical significance was determined by unpaired students two-tailed t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns not significant. Source data are provided as a Source Data file.