PHILOSOPHICAL TRANSACTIONS B rstb.royalsocietypublishing.org Review CrossMark click for updates Cite this article: Hille F, Charpentier E. 2016 CRISPR-Cas: biology, mechanisms and relevance. Phil. Trans. R. Soc. B 371: 20150496. http://dx.doi.org/10.1098/rstb.2015.0496 Accepted: 10 August 2016 One contribution of 15 to a discussion meeting issue 'The new bacteriology'. Subject Areas: microbiology, biochemistry, evolution, genetics, molecular biology Keywords: CRISPR, Cas9, bacteriophage, genome editing Author for correspondence: Emmanuelle Charpentier e-mail: charpentier@mpiib-berlin.mpg.de CRISPR-Cas: biology, mechanisms and relevance Frank Hille1 and Emmanuelle Charpentier1,2 1Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin 10117, Germany 2The Laboratory for Molecular Infection Medicine Sweden (MIMS), UmeaCentre for Microbial Research (UCMR), Department of Molecular Biology, Umea University, Umeå 90187, Sweden ?D EC, 0000-0002-0254-0778 Prokaryotes have evolved several defence mechanisms to protect themselves from viral predators. Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) display a prokaryotic adaptive immune system that memorizes previous infections by integrating short sequences of invading genomes-termed spacers-into the CRISPR locus. The spacers interspaced with repeats are expressed as small guide CRISPR RNAs (crRNAs) that are employed by Cas proteins to target inva- ders sequence-specifically upon a reoccurring infection. The ability of the minimal CRISPR-Cas9 system to target DNA sequences using programma- ble RNAs has opened new avenues in genome editing in a broad range of cells and organisms with high potential in therapeutical applications. While numerous scientific studies have shed light on the biochemical pro- cesses behind CRISPR-Cas systems, several aspects of the immunity steps, however, still lack sufficient understanding. This review summarizes major discoveries in the CRISPR-Cas field, discusses the role of CRISPR- Cas in prokaryotic immunity and other physiological properties, and describes applications of the system as a DNA editing technology and antimicrobial agent. This article is part of the themed issue 'The new bacteriology'. 1. Introduction Being the most abundant entities on our planet, bacterial and archaeal viruses (bacteriophages or phages) display a constant threat to prokaryotic life. In order to withstand phages, prokaryotes have evolved several defence strategies. In the past decade, the prokaryotic immune system CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) caught increasing attention in the scientific community not only because of its unique adaptive nature, but also because of its therapeutic potential. This review seeks to sum- marize the major discoveries made in the field of CRISPR-Cas, and describes the biological roles of the system in antiviral defence and other biological pathways as well as its significance for medical application. CRISPR-Cas is the only adaptive immune system in prokaryotes known so far. In this system, small guide RNAs (crRNAs) are employed for sequence- specific interference with invading nucleic acids. CRISPR-Cas comprises a genomic locus called CRISPR that harbours short repetitive elements (repeats) separated by unique sequences (spacers), which can originate from mobile genetic elements (MGEs) such as bacteriophages, transposons or plasmids. The so-called CRISPR array is preceded by an AT-rich leader sequence and is usually flanked by a set of cas genes encoding the Cas proteins [1-4]. To date, CRISPR-Cas systems