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Non-specific degradation of transcripts promotes plasmid clearance during type III-A CRISPR–Cas immunity


Type III-A CRISPR–Cas systems employ the Cas10–Csm complex to destroy bacteriophages and plasmids, using a guide RNA to locate complementary RNA molecules from the invader and trigger an immune response that eliminates the infecting DNA. In addition, these systems possess the non-specific RNase Csm6, which provides further protection for the host. While the role of Csm6 in immunity during phage infection has been determined, how this RNase is used against plasmids is unclear. Here, we show that Staphylococcus epidermidis Csm6 is required for immunity when transcription across the plasmid target is infrequent, leading to impaired target recognition and inefficient DNA degradation by the Cas10–Csm complex. In these conditions, Csm6 causes growth arrest in the host and prevents further plasmid replication through the indiscriminate degradation of host and plasmid transcripts. In contrast, when plasmid target sequences are efficiently transcribed, Csm6 is dispensable and DNA degradation by Cas10 is sufficient for anti-plasmid immunity. Csm6 therefore provides robustness to the type III-A CRISPR–Cas immune response against difficult targets for the Cas10–Csm complex.

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Data availability

The data from this study are available from the authors upon request. The raw data for the RNA-seq experiments can be found at the Sequence Read Archive (NIH) through accession code PRJNA506073. Original gel pictures and northern blots are provided in Supplementary Fig. 6.

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The authors would like to thank A. Meeske and C. Mo for critical reading of the manuscript. They also thank the following: G. Goldberg for plasmid pGG25; C. Kenney and W. Jiang for sharing their insights on spc1-flip conjugation; the Rockefeller University Genomics Resource Center for performing the Csm6-targeting Nextseq RNA-seq experiment; and T. Carroll (of the Rockefeller University Bioinformatics Resource Center) and E. Stoyanova for helpful discussions on the bioinformatic analysis. J.T.R. was supported by a Boehringer Ingelheim Fonds Ph.D. fellowship. L.M. is supported by a Burroughs Wellcome Fund PATH Award and a NIH Director’s Pioneer Award (DP1GM128184).

Author information

J.T.R. and L.M. designed the study. J.T.R performed all experiments and analysed the next-generation sequencing data. J.T.R. and L.M. wrote the manuscript.

Competing interests

L.M. is a cofounder and Scientific Advisory Board member of Intellia Therapeutics and a cofounder of Eligo Biosciences. J.T.R. declares no competing interests.

Correspondence to Luciano A. Marraffini.

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    Supplementary Figures 1–6, Supplementary Tables 1–3 and Supplementary References.

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Fig. 1: Csm6 is required for interference against pG0400 when the target is weakly transcribed.
Fig. 2: Csm6 accelerates plasmid clearance when interfering against a weakly transcribed protospacer.
Fig. 3: Csm6 activation results in non-specific degradation of host and plasmid transcripts.
Fig. 4: Prevention of expression of genes important for plasmid replication accelerates plasmid clearance.
Fig. 5: The Cas10 HD domain is required for efficient plasmid clearance during type III-A immunity.