Fig. 6
Loss of plasmid clearance linked to low crRNA abundance and global RNA secondary structure. a Multiplexed plasmid clearance by FnCas12a in E. coli. See Fig. 3a for details. Sequences of the spacers and junctions in the assembled arrays are shown in Supplementary Table 4. b RNA-seq analysis of the three-spacer arrays in PcF-2/3/1 and PcF-1/3/2. Each array and FnCas12a were co-expressed in TXTL prior to small-RNA isolation and RNA-seq analysis. See Fig. 5 for details. Stars indicate mapped reads for spacer S1. c Northern blotting analysis of the transcribed arrays from PcF-2/3/1 and PcF-3/2/1. Each array and FnCas12a were co-expressed in E. coli prior to total RNA isolation and Northern blotting analysis. The region in which the processed crRNAs should appear is shown with greater intensity. The analysis was conducted with a radiolabeled oligo probe complementary to spacer S1. Detection of 5S RNA was used as a loading control. Gel images from an independent experiment are in Supplementary Fig. 7. d Mutations made within a predicted imperfect hairpin between spacers S1 and S3. The predicted hairpin was present as a stable structure (base-pairing probabilities > 80%) only in PcF-2/3/1. Note that the structure prevents formation of the hairpin recognized by FnCas12a (gray dashed line). One set of mutations (m1– m5) were selected to disrupt the predicted secondary structure. Another set (m4’, m5’) represents expanded mutations to m4 and m5 that reform a stable secondary structure. Predictions of the minimal-free energy structure and base-pairing probabilities were made for the sequence spanning the 5′ repeat through the 3′ spacer. Text is colored to match that of spacer S3 (green), spacer S1 (blue), the assembly junctions (brown), and the intervening repeat (black). Mutations are in red. e Plasmid clearance directed by FnCas12a and the mutated versions of PcF-2/3/1. The protospacer in the target plasmid was mutated to match each changed spacer sequence. See Fig. 3a for details. Sequences of the spacers and junctions in the assembled arrays are shown in Supplementary Table 4. Values represent the geometric mean and S.D. from three independent transformations starting from separate colonies. Source data are provided as a Source Data file
