title: Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems.
abstract: The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA:crRNA duplex (dual-RNA), which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci. The reported collection of dual-RNA:Cas9 with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9 tool.
authors.affiliation: The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå Centre for Microbial Research (UCMR), Department of Molecular Biology, Umeå University, Umeå S-90187, Sweden, Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig D-38124, Germany, Deptartment of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, Vienna A-1030, Austria, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA and Hannover Medical School, Hannover D-30625, Germany.
authors.forename: Ines
authors.initials: I
authors.lastname: Fonfara
authors.forename: Anaïs
authors.initials: A
authors.lastname: Le Rhun
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Chylinski
authors.forename: Kira S
authors.initials: KS
authors.lastname: Makarova
authors.forename: Anne-Laure
authors.initials: AL
authors.lastname: Lécrivain
authors.forename: Janek
authors.initials: J
authors.lastname: Bzdrenga
authors.forename: Eugene V
authors.initials: EV
authors.lastname: Koonin
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Charpentier
mesh_terms: D001419:Bacteria / Q000201:enzymology; D064130:CRISPR-Associated Proteins / Q000737:chemistry / Q000145:classification* / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D053837:DNA Cleavage; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000145:classification* / Q000378:metabolism*; D059372:Nucleotide Motifs; D010802:Phylogeny; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D043244:Ribonuclease III / Q000378:metabolism; D013297:Streptococcus pyogenes / Q000201:enzymology
chemical_list: D064130:CRISPR-Associated Proteins; D012313:RNA; D004247:DNA; D004706:Endodeoxyribonucleases; D043244:Ribonuclease III