Crystal Structure of the Minimal Cas9 from Campylobacter jejuni Reveals the Molecular Diversity in the CRISPR-Cas9 Systems.

The RNA-guided endonuclease Cas9 generates a double-strand break at DNA target sites complementary to the guide RNA and has been harnessed for the development of a variety of new technologies, such as genome editing. Here, we report the crystal structures of Campylobacter jejuni Cas9 (CjCas9), one of the smallest Cas9 orthologs, in complex with an sgRNA and its target DNA. The structures provided insights into a minimal Cas9 scaffold and revealed the remarkable mechanistic diversity of the CRISPR-Cas9 systems. The CjCas9 guide RNA contains a triple-helix structure, which is distinct from known RNA triple helices, thereby expanding the natural repertoire of RNA triple helices. Furthermore, unlike the other Cas9 orthologs, CjCas9 contacts the nucleotide sequences in both the target and non-target DNA strands and recognizes the 5'-NNNVRYM-3' as the protospacer-adjacent motif. Collectively, these findings improve our mechanistic understanding of the CRISPR-Cas9 systems and may facilitate Cas9 engineering.

[1]  J. Steitz,et al.  Structural insights into the stabilization of MALAT1 noncoding RNA by a bipartite triple helix , 2014, Nature Structural &Molecular Biology.

[2]  Adam M. Phillippy,et al.  Interactive metagenomic visualization in a Web browser , 2011, BMC Bioinformatics.

[3]  Kira S. Makarova,et al.  Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems , 2016, Science.

[4]  Tautvydas Karvelis,et al.  Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements , 2015, Genome Biology.

[5]  A. Regev,et al.  Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System , 2015, Cell.

[6]  R. Barrangou,et al.  Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria , 2012, Proceedings of the National Academy of Sciences.

[7]  C. A. Theimer,et al.  Structure of the human telomerase RNA pseudoknot reveals conserved tertiary interactions essential for function. , 2005, Molecular cell.

[8]  J. García-Martínez,et al.  Short motif sequences determine the targets of the prokaryotic CRISPR defence system. , 2009, Microbiology.

[9]  Jennifer A. Doudna,et al.  Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage , 2016, Science.

[10]  Jennifer A. Doudna,et al.  Conformational control of DNA target cleavage by CRISPR–Cas9 , 2015, Nature.

[11]  R. Batey,et al.  Structure of the SAM-II riboswitch bound to S-adenosylmethionine , 2008, Nature Structural &Molecular Biology.

[12]  J. Doudna,et al.  A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity , 2012, Science.

[13]  H. Nishimasu,et al.  Structures and mechanisms of CRISPR RNA-guided effector nucleases. , 2017, Current opinion in structural biology.

[14]  Feng Zhang,et al.  Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA , 2014, Cell.

[15]  Eric Westhof,et al.  The non-Watson-Crick base pairs and their associated isostericity matrices. , 2002, Nucleic acids research.

[16]  Yan Zhang,et al.  DNase H Activity of Neisseria meningitidis Cas9. , 2015, Molecular cell.

[17]  Philippe Horvath,et al.  The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA , 2010, Nature.

[18]  Luciano A. Marraffini,et al.  CRISPR-Cas immunity in prokaryotes , 2015, Nature.

[19]  Randy J. Read,et al.  Acta Crystallographica Section D Biological , 2003 .

[20]  R. Sinsheimer.  Single-stranded DNA. , 1962, Scientific American.

[21]  Takanori Nakane,et al.  Structure and Engineering of Francisella novicida Cas9 , 2016, Cell.

[22]  Lucas B. Harrington,et al.  Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes. , 2015, Molecular cell.

[23]  Philip R. Evans,et al.  How good are my data and what is the resolution? , 2013, Acta crystallographica. Section D, Biological crystallography.

[24]  J. Vogel,et al.  CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III , 2011, Nature.

[25]  David A. Scott,et al.  In vivo genome editing using Staphylococcus aureus Cas9 , 2015, Nature.

[26]  M. Jinek,et al.  Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease , 2014, Nature.

[27]  G. Crooks,et al.  WebLogo: a sequence logo generator. , 2004, Genome research.

[28]  Emmanuelle Charpentier,et al.  The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems , 2013, RNA biology.

[29]  Chase L. Beisel,et al.  Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems. , 2016, Molecular cell.

[30]  Philippe Horvath,et al.  Phage Response to CRISPR-Encoded Resistance in Streptococcus thermophilus , 2007, Journal of bacteriology.

[31]  Sita J. Saunders,et al.  An updated evolutionary classification of CRISPR–Cas systems , 2015, Nature Reviews Microbiology.

[32]  Kevin Cowtan,et al.  The Buccaneer software for automated model building. 1. Tracing protein chains. , 2006, Acta crystallographica. Section D, Biological crystallography.

[33]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[34]  Le Cong,et al.  Multiplex Genome Engineering Using CRISPR/Cas Systems , 2013, Science.

[35]  Yinqing Li,et al.  Crystal Structure of Staphylococcus aureus Cas9 , 2015, Cell.

[36]  Jennifer Doudna,et al.  RNA-programmed genome editing in human cells , 2013, eLife.

[37]  R. Barrangou,et al.  Applications of CRISPR technologies in research and beyond , 2016, Nature Biotechnology.

[38]  Jennifer A. Doudna,et al.  Biology and Applications of CRISPR Systems: Harnessing Nature’s Toolbox for Genome Engineering , 2016, Cell.

[39]  Kira S. Makarova,et al.  Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems , 2013, Nucleic acids research.

[40]  E. Lander,et al.  Development and Applications of CRISPR-Cas9 for Genome Engineering , 2014, Cell.

[41]  James E. DiCarlo,et al.  RNA-Guided Human Genome Engineering via Cas9 , 2013, Science.

[42]  Jennifer A. Doudna,et al.  A Cas9–guide RNA complex preorganized for target DNA recognition , 2015, Science.

[43]  Nicholas K. Sauter,et al.  The DIALS framework for integration software , 2013 .

[44]  Jennifer A. Doudna,et al.  Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation , 2014, Science.