Fig. 4. SuCas12a2 causes abortive infection principally through collateral DNA degradation and can be harnessed for RNA detection.

a, E. coli growth arrest in the presence of SuCas12a2, LbCas2a2 or LsCas13a plasmid under different targeting conditions and antibiotic regimes. A₆₀₀, absorbance at 600 nm. b, The percentage of E. coli cells stained with propidium iodide (PI) indicative of viability loss before targeting (uninduced) and after 4 h of targeting (induced) without antibiotic selection. The gating strategy is shown in Supplementary Fig. 7. c, The varying extent of RNA degradation in E. coli by SuCas12a2, LbCas12a or LsCas13a2 2 h after induction without antibiotic selection. The results represent duplicate independent experiments. See Extended Data Fig. 7 for independent quadruplicates. The small RNA pool includes tRNAs and other small RNAs. d, SOS-responsive expression of GFP in E. coli after 4 h of plasmid targeting by SuCas12a2, LbCas12a or LsCas13a without antibiotic selection. Time-course data are shown in Supplementary Fig. 9. RFU, relative fluorescence units. e, Relative DNA content in E. coli after 4 h of targeting by SuCas12a2, LbCas12a or LsCas13a without antibiotic selection. 4′,6-diamidino-2-phenylindole (DAPI) fluorescence and cell size were measured using flow cytometry. Each circle or pair of vertically aligned circles represents major subpopulations from the same biological replicate. The corresponding contour plots are shown in Extended Data Fig. 8. FSC, forward scatter. f, RNA-detection assay. The limit of RNA detection with Cas12a2 incubated with a ssRNA beacon at room temperature (RT). Data are mean ± s.e.m. of three independent experiments. g, Nucleic-acid targets and reporters as well as the unamplified limit of detection (LOD) for Cas12a2 and other Cas detectors⁴⁶. Aap, Alicyclobacillus acidiphilus; Lwa, Leptotrichia wadei; Lbu, Leptotrichia buccalis. h, The proposed model for promiscuous RNA targeting and collateral degradation by SuCas12a2 and its effect on the cell. For b and d, data are mean ± s.d. of four independent experiments started from separate colonies. Statistical analysis was performed using one-tailed Welch’s t-tests. NT, non-target plasmid; T, target plasmid.