Co-transcriptional DNA and RNA Cleavage during Type III CRISPR-Cas Immunity

Samai, Poulami; Pyenson, Nora C.; Jiang, Wenyan; Goldberg, Gregory W.; Hatoum-Aslan, Asma; Marraffini, Luciano A.

doi:10.1016/j.cell.2015.04.027

Cited by 397 publications

(550 citation statements)

References 41 publications

Supporting

Mentioning

522

Contrasting

Order By: Relevance

“…No difference was observed (Fig. 3), in agreement with the current view that type IIIA and most likely also type IIIB complexes strictly require transcription of their DNA substrates (Samai et al, 2015).…”

Section: Activity Assaysupporting

confidence: 80%

“…The puzzle was solved by the observation that plasmid immunity of Sulfolobus islandicus REY15A (a host of two different Cmr complexes) required transcription (Deng et al, 2013). Subsequently, it was shown that the Csm complex from Staphylococcus epidermidis also harbored a strictly transcription dependent DNA endonuclease activity (Samai et al, 2015). As the DNA and RNA endonuclease active sites were distinct, their roles could be genetically separated.…”

Section: Introductionmentioning

confidence: 99%

“…As the DNA and RNA endonuclease active sites were distinct, their roles could be genetically separated. Again using the S. epidermidis model, it was shown that the major contribution to plasmid and (DNA) virus immunity was made by the DNA endonucleolytic activity, with RNA endonucleolytic activity rather serving as a "backup" (Samai et al, 2015). Proto-spacer adjacent motifs are not required in targets of type III CRISPR systems.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Type III CRISPR complexes from Thermus thermophilus.

et al. 2016

View full text Add to dashboard Cite

Pathogen-specific acquired immunity in bacteria is mediated by the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems. Thermus thermophilus strain HB8 contains CRISPR systems of several major subtypes (type I, IIIA and IIIB), and has become a widely studied model for CRISPR biology. We have selected two highly expressed CRISPR spacers, crRNA 2.1 and crRNA 2.2, and have enriched endogenous T. thermophilus proteins that co-purify with these crRNAs. Mass spectroscopy indicates that the chromatography protocol enriches predominantly Csm complex subunits, but also Cmr subunits. After several chromatographic steps, size exclusion chromatography indicated a molecular mass of the crRNA associated complex of 265±69 kDa. In agreement with earlier work, crRNAs of different lengths (containing the selected spacers) were observed. Most of these were completely lost when several T. thermophilus csm genes were ablated.

show abstract

Section: Activity Assaysupporting

confidence: 80%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Type III CRISPR complexes from Thermus thermophilus.

et al. 2016

View full text Add to dashboard Cite

show abstract

“…Purified Csm complexes from Streptococcus thermophilus and Thermus thermophilus cleave ssRNA in vitro by a mechanism similar to that of the Cmr complex (Staals et al 2014;Tamulaitis et al 2014). The S. epidermidis Csm complex has recently been shown to harbor two independent endonuclease activities (Samai et al 2015). The complex is capable of cleaving ssRNA targets complementary to the crRNA; additionally, the complex cleaves double-stranded target DNAs within the nontemplate strand in a transcription-dependent manner.…”

Section: Introductionmentioning

confidence: 99%

Structural basis for the endoribonuclease activity of the type III-A CRISPR-associated protein Csm6

Niewoehner

Jinek

2016

RNA

135

134

View full text Add to dashboard Cite

Prokaryotic CRISPR-Cas systems provide an RNA-guided mechanism for genome defense against mobile genetic elements such as viruses and plasmids. In type III-A CRISPR-Cas systems, the RNA-guided multisubunit Csm effector complex targets both singlestranded RNAs and double-stranded DNAs. In addition to the Csm complex, efficient anti-plasmid immunity mediated by type III-A systems also requires the CRISPR-associated protein Csm6. Here we report the crystal structure of Csm6 from Thermus thermophilus and show that the protein is a ssRNA-specific endoribonuclease. The structure reveals a dimeric architecture generated by interactions involving the N-terminal CARF and C-terminal HEPN domains. HEPN domain dimerization leads to the formation of a composite ribonuclease active site. Consistently, mutations of invariant active site residues impair catalytic activity in vitro. We further show that the ribonuclease activity of Csm6 is conserved across orthologs, suggesting that it plays an important functional role in CRISPR-Cas systems. The dimer interface of the CARF domains features a conserved electropositive pocket that may function as a ligand-binding site for allosteric control of ribonuclease activity. Altogether, our work suggests that Csm6 proteins provide an auxiliary RNA-targeting interference mechanism in type III-A CRISPR-Cas systems that operates in conjunction with the RNA-and DNA-targeting endonuclease activities of the Csm effector complex.

show abstract

“…Type III systems lack Cas3, and the protein responsible for target cleavage is Cas10, which contains polymerase-cyclase and HD-nuclease domains that are both required for the target degradation (31,32).…”

mentioning

confidence: 99%

Recruitment of CRISPR-Cas systems by Tn7-like transposons

Peters

Makarova

Shmakov

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

245

267

View full text Add to dashboard Cite

A survey of bacterial and archaeal genomes shows that many Tn7-like transposons contain minimal type I-F CRISPR-Cas systems that consist of fused cas8f and cas5f, cas7f, and cas6f genes and a short CRISPR array. Several small groups of Tn7-like transposons encompass similarly truncated type I-B CRISPR-Cas. This minimal gene complement of the transposon-associated CRISPR-Cas systems implies that they are competent for pre-CRISPR RNA (precrRNA) processing yielding mature crRNAs and target binding but not target cleavage that is required for interference. Phylogenetic analysis demonstrates that evolution of the CRISPR-Cas-containing transposons included a single, ancestral capture of a type I-F locus and two independent instances of type I-B loci capture. We show that the transposon-associated CRISPR arrays contain spacers homologous to plasmid and temperate phage sequences and, in some cases, chromosomal sequences adjacent to the transposon. We hypothesize that the transposon-encoded CRISPR-Cas systems generate displacement (R-loops) in the cognate DNA sites, targeting the transposon to these sites and thus facilitating their spread via plasmids and phages. These findings suggest the existence of RNAguided transposition and fit the guns-for-hire concept whereby mobile genetic elements capture host defense systems and repurpose them for different stages in the life cycle of the element.CRISPR-Cas systems | Tn7 transposon | transposition strategy | crRNA guide | target-site selection

show abstract

Co-transcriptional DNA and RNA Cleavage during Type III CRISPR-Cas Immunity

Cited by 397 publications

References 41 publications

Type III CRISPR complexes from Thermus thermophilus.

Type III CRISPR complexes from Thermus thermophilus.

Structural basis for the endoribonuclease activity of the type III-A CRISPR-associated protein Csm6

Recruitment of CRISPR-Cas systems by Tn7-like transposons

Contact Info

Product

Resources

About