CRISPR RNA-guided DNA cleavage by reconstituted Type I-A immune effector complexes
Diverse CRISPR-Cas immune systems protect archaea and bacteria from viruses and other mobile genetic elements. All CRISPR-Cas systems ultimately function by sequence-specific destruction of invading complementary nucleic acids. However, each CRISPR system uses compositionally distinct crRNP [CRISPR (cr) RNA/Cas protein] immune effector complexes to recognize and destroy invasive nucleic acids by unique molecular mechanisms. Previously, we found that Type I-A (Csa) effector crRNPs from Pyrococcus furiosus function in vivo to eliminate invader DNA. Here, we reconstituted functional Type I-A effector crRNPs in vitro with recombinant Csa proteins and synthetic crRNA and characterized properties of crRNP assembly, target DNA recognition and cleavage. Six proteins (Csa 4-1, Cas3″, Cas3′, Cas5a, Csa2, Csa5) are essential for selective target DNA binding and cleavage. Native gel shift analysis and UV-induced RNA–protein crosslinking demonstrate that Cas5a and Csa2 directly interact with crRNA 5′ tag and guide sequences, respectively. Mutational analysis revealed that Cas3″ is the effector nuclease of the complex. Together, our results indicate that DNA cleavage by Type I-A crRNPs requires crRNA-guided and protospacer adjacent motif-dependent target DNA binding to unwind double-stranded DNA and expose single strands for progressive ATP-dependent 3′–5′ cleavage catalyzed by integral Cas3′ helicase and Cas3″ nuclease crRNP components.
KeywordsCRISPR Cas Csa Cas3 Type I-A Pyrococcus furiosus
We thank Rebecca Terns for valuable mentorship, discussions and early contributions to the writing of this manuscript. We are also grateful to members of Terns’ lab for their technical input, Claiborne V. C. Glover III for critical reading of the manuscript, and Dr. Hong Li (Florida State University) for contributing purified Cas3″ and Cas3′ proteins shown in Fig. S5b. This work was supported by National Institutes of Health grant R35GM118160 to M.P.T.
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