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Structural basis for promiscuous PAM recognition in type I-E Cascade from E. coli.

Hayes RP, Xiao Y, Ding F, van Erp PB, Rajashankar K, Bailey S, Wiedenheft B, Ke A
Nature 530 499-503 (2016)
Cited: 103 times
EuropePMC logo PMID: 26863189

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPRs) and the cas (CRISPR-associated) operon form an RNA-based adaptive immune system against foreign genetic elements in prokaryotes. Type I accounts for 95% of CRISPR systems, and has been used to control gene expression and cell fate. During CRISPR RNA (crRNA)-guided interference, Cascade (CRISPR-associated complex for antiviral defence) facilitates the crRNA-guided invasion of double-stranded DNA for complementary base-pairing with the target DNA strand while displacing the non-target strand, forming an R-loop. Cas3, which has nuclease and helicase activities, is subsequently recruited to degrade two DNA strands. A protospacer adjacent motif (PAM) sequence flanking target DNA is crucial for self versus foreign discrimination. Here we present the 2.45 Å crystal structure of Escherichia coli Cascade bound to a foreign double-stranded DNA target. The 5'-ATG PAM is recognized in duplex form, from the minor groove side, by three structural features in the Cascade Cse1 subunit. The promiscuity inherent to minor groove DNA recognition rationalizes the observation that a single Cascade complex can respond to several distinct PAM sequences. Optimal PAM recognition coincides with wedge insertion, initiating directional target DNA strand unwinding to allow segmented base-pairing with crRNA. The non-target strand is guided along a parallel path 25 Å apart, and the R-loop structure is further stabilized by locking this strand behind the Cse2 dimer. These observations provide the structural basis for understanding the PAM-dependent directional R-loop formation process.

Articles - 5h9e mentioned but not cited (5)

  1. Structural basis for promiscuous PAM recognition in type I-E Cascade from E. coli. Hayes RP, Xiao Y, Ding F, van Erp PB, Rajashankar K, Bailey S, Wiedenheft B, Ke A. Nature 530 499-503 (2016)
  2. Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System. Xiao Y, Luo M, Hayes RP, Kim J, Ng S, Ding F, Liao M, Ke A. Cell 170 48-60.e11 (2017)
  3. PAM identification by CRISPR-Cas effector complexes: diversified mechanisms and structures. Gleditzsch D, Pausch P, Müller-Esparza H, Özcan A, Guo X, Bange G, Randau L. RNA Biol 16 504-517 (2019)
  4. Role of nucleotide identity in effective CRISPR target escape mutations. Künne T, Zhu Y, da Silva F, Konstantinides N, McKenzie RE, Jackson RN, Brouns SJ. Nucleic Acids Res 46 10395-10404 (2018)
  5. Bioinformatics comparisons of RNA-binding proteins of pathogenic and non-pathogenic Escherichia coli strains reveal novel virulence factors. Ghosh P, Sowdhamini R. BMC Genomics 18 658 (2017)


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  2. The next generation of CRISPR-Cas technologies and applications. Pickar-Oliver A, Gersbach CA. Nat Rev Mol Cell Biol 20 490-507 (2019)
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  11. Chemistry of Class 1 CRISPR-Cas effectors: Binding, editing, and regulation. Liu TY, Doudna JA. J Biol Chem 295 14473-14487 (2020)
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  16. Harnessing "A Billion Years of Experimentation": The Ongoing Exploration and Exploitation of CRISPR-Cas Immune Systems. Klompe SE, Sternberg SH. CRISPR J 1 141-158 (2018)
  17. Expanding the plant genome editing toolbox with recently developed CRISPR-Cas systems. Wada N, Osakabe K, Osakabe Y. Plant Physiol 188 1825-1837 (2022)
  18. CRISPR-Enabled Tools for Engineering Microbial Genomes and Phenotypes. Tarasava K, Oh EJ, Eckert CA, Gill RT. Biotechnol J 13 e1700586 (2018)
  19. Mechanisms of Type I-E and I-F CRISPR-Cas Systems in Enterobacteriaceae. Xue C, Sashital DG. EcoSal Plus 8 (2019)
  20. Creating memories: molecular mechanisms of CRISPR adaptation. Lee H, Sashital DG. Trends Biochem Sci 47 464-476 (2022)
  21. Digging into the lesser-known aspects of CRISPR biology. Guzmán NM, Esquerra-Ruvira B, Mojica FJM. Int Microbiol 24 473-498 (2021)
  22. Single-Molecule View of Small RNA-Guided Target Search and Recognition. Globyte V, Kim SH, Joo C. Annu Rev Biophys 47 569-593 (2018)
  23. Progress and Application of CRISPR/Cas Technology in Biological and Biomedical Investigation. Lin J, Zhou Y, Liu J, Chen J, Chen W, Zhao S, Wu Z, Wu N. J Cell Biochem 118 3061-3071 (2017)
  24. Advances in the application of CRISPR-Cas technology in rapid detection of pathogen nucleic acid. Li X, Zhong J, Li H, Qiao Y, Mao X, Fan H, Zhong Y, Imani S, Zheng S, Li J. Front Mol Biosci 10 1260883 (2023)
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  26. The biology and type I/III hybrid nature of type I-D CRISPR-Cas systems. McBride TM, Cameron SC, Fineran PC, Fagerlund RD. Biochem J 480 471-488 (2023)
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