PAM-Dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease

Overview

Journal Cell

Publisher Cell Press

Specialty Cell Biology

Date 2016 Dec 17

PMID 27984729

Citations 124

Authors

Hui Yang

Pu Gao

Kanagalaghatta R Rajashankar

Dinshaw J Patel

Affiliations

Soon will be listed here.

Abstract

C2c1 is a newly identified guide RNA-mediated type V-B CRISPR-Cas endonuclease that site-specifically targets and cleaves both strands of target DNA. We have determined crystal structures of Alicyclobacillus acidoterrestris C2c1 (AacC2c1) bound to sgRNA as a binary complex and to target DNAs as ternary complexes, thereby capturing catalytically competent conformations of AacC2c1 with both target and non-target DNA strands independently positioned within a single RuvC catalytic pocket. Moreover, C2c1-mediated cleavage results in a staggered seven-nucleotide break of target DNA. crRNA adopts a pre-ordered five-nucleotide A-form seed sequence in the binary complex, with release of an inserted tryptophan, facilitating zippering up of 20-bp guide RNA:target DNA heteroduplex on ternary complex formation. Notably, the PAM-interacting cleft adopts a "locked" conformation on ternary complex formation. Structural comparison of C2c1 ternary complexes with their Cas9 and Cpf1 counterparts highlights the diverse mechanisms adopted by these distinct CRISPR-Cas systems, thereby broadening and enhancing their applicability as genome editing tools.

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References

Nakanishi K, Weinberg D, Bartel D, Patel D . Structure of yeast Argonaute with guide RNA. Nature. 2012; 486(7403):368-74. PMC: 3853139. DOI: 10.1038/nature11211. View

Deveau H, Barrangou R, Garneau J, Labonte J, Fremaux C, Boyaval P . Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J Bacteriol. 2007; 190(4):1390-400. PMC: 2238228. DOI: 10.1128/JB.01412-07. View

van der Oost J, Jore M, Westra E, Lundgren M, Brouns S . CRISPR-based adaptive and heritable immunity in prokaryotes. Trends Biochem Sci. 2009; 34(8):401-7. DOI: 10.1016/j.tibs.2009.05.002. View

Emsley P, Lohkamp B, Scott W, Cowtan K . Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 2010; 66(Pt 4):486-501. PMC: 2852313. DOI: 10.1107/S0907444910007493. View

Shmakov S, Abudayyeh O, Makarova K, Wolf Y, Gootenberg J, Semenova E . Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems. Mol Cell. 2015; 60(3):385-97. PMC: 4660269. DOI: 10.1016/j.molcel.2015.10.008. View

Makarova K, Wolf Y, Alkhnbashi O, Costa F, Shah S, Saunders S . An updated evolutionary classification of CRISPR-Cas systems. Nat Rev Microbiol. 2015; 13(11):722-36. PMC: 5426118. DOI: 10.1038/nrmicro3569. View

Deltcheva E, Chylinski K, Sharma C, Gonzales K, Chao Y, Pirzada Z . CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 2011; 471(7340):602-7. PMC: 3070239. DOI: 10.1038/nature09886. View

Gao P, Yang H, Rajashankar K, Huang Z, Patel D . Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition. Cell Res. 2016; 26(8):901-13. PMC: 4973337. DOI: 10.1038/cr.2016.88. View

Sternberg S, Doudna J . Expanding the Biologist's Toolkit with CRISPR-Cas9. Mol Cell. 2015; 58(4):568-74. DOI: 10.1016/j.molcel.2015.02.032. View

10.

Dupuis M, Villion M, Magadan A, Moineau S . CRISPR-Cas and restriction-modification systems are compatible and increase phage resistance. Nat Commun. 2013; 4:2087. DOI: 10.1038/ncomms3087. View

11.

Hille F, Charpentier E . CRISPR-Cas: biology, mechanisms and relevance. Philos Trans R Soc Lond B Biol Sci. 2016; 371(1707). PMC: 5052741. DOI: 10.1098/rstb.2015.0496. View

12.

Elkayam E, Kuhn C, Tocilj A, Haase A, Greene E, Hannon G . The structure of human argonaute-2 in complex with miR-20a. Cell. 2012; 150(1):100-10. PMC: 3464090. DOI: 10.1016/j.cell.2012.05.017. View

13.

Nishimasu H, Ran F, Hsu P, Konermann S, Shehata S, Dohmae N . Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014; 156(5):935-49. PMC: 4139937. DOI: 10.1016/j.cell.2014.02.001. View

14.

Gasiunas G, Barrangou R, Horvath P, Siksnys V . Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A. 2012; 109(39):E2579-86. PMC: 3465414. DOI: 10.1073/pnas.1208507109. View

15.

Mojica F, Diez-Villasenor C, Garcia-Martinez J, Almendros C . Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology (Reading). 2009; 155(Pt 3):733-740. DOI: 10.1099/mic.0.023960-0. View

16.

Yamano T, Nishimasu H, Zetsche B, Hirano H, Slaymaker I, Li Y . Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA. Cell. 2016; 165(4):949-62. PMC: 4899970. DOI: 10.1016/j.cell.2016.04.003. View

17.

Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S . CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007; 315(5819):1709-12. DOI: 10.1126/science.1138140. View

18.

Jiang F, Zhou K, Ma L, Gressel S, Doudna J . STRUCTURAL BIOLOGY. A Cas9-guide RNA complex preorganized for target DNA recognition. Science. 2015; 348(6242):1477-81. DOI: 10.1126/science.aab1452. View

19.

Adams P, Afonine P, Bunkoczi G, Chen V, Davis I, Echols N . PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr. 2010; 66(Pt 2):213-21. PMC: 2815670. DOI: 10.1107/S0907444909052925. View

20.

Anders C, Niewoehner O, Duerst A, Jinek M . Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature. 2014; 513(7519):569-73. PMC: 4176945. DOI: 10.1038/nature13579. View