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. Author manuscript; available in PMC: 2020 Jun 9.
Published in final edited form as: Nature. 2019 Dec 9;577(7789):244–248. doi: 10.1038/s41586-019-1786-y

Figure 4: Phage ΦKZ DNA is sensitive to RNA-targeting Cas13.

Figure 4:

a, PAO1 expressing LseCas13a and a crRNA targeting phage ΦKZ. Plaque assays were conducted as in Fig. 1a. b, Growth curves measuring the OD600 of PAO1 infected with the indicated ΦKZ MOI. c, Cas13 escaper ΦKZ phage mutant deletions (red dashed lines) with target sites indicated (blue line). d, Live fluorescence imaging of P. aeruginosa strains engineered to express LseCas13a and crRNAs targeting ΦKZ. Cyan stain shows the phage DNA. e, Model summarizing the ΦKZ nucleus-like structure excluding (flat arrow) Cas9, Cas12, Cascade-Cas3 (Type I-C, Type I-F) and Type I restriction endonucleases (REase) and Type II REase, while the mRNA (red) is exported and can be targeted by Cas13. The nucleus-like structure is resistant to the indicated nucleases, but EcoRI fusion (to internal protein ORF152) enables targeting. Cas13 plaque assays a were replicated >3 times with similar results. Growth curve experiments b were replicated twice with similar results. Escaper mutants c were isolated once and verified by PCR, sequencing, and plaque assays. Microscopy d was replicated twice with similar results.