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CRISPR-Cas: Biology, Mechanisms and Relevance


CRISPR-Cas: Biology, Mechanisms and Relevance

Frank Hille et al. Philos Trans R Soc Lond B Biol Sci.


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 invaders sequence-specifically upon a reoccurring infection. The ability of the minimal CRISPR-Cas9 system to target DNA sequences using programmable 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 processes 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'.

Keywords: CRISPR; Cas9; bacteriophage; genome editing.


Figure 1.
Figure 1.
Simplified model of the immunity mechanisms of class 1 and class 2 CRISPR-Cas systems. The CRISPR-Cas systems are composed of a cas operon (blue arrows) and a CRISPR array that comprises identical repeat sequences (black rectangles) that are interspersed by phage-derived spacers (coloured rectangles). Upon phage infection, a sequence of the invading DNA (protospacer) is incorporated into the CRISPR array by the Cas1–Cas2 complex. The CRISPR array is then transcribed into a long precursor CRISPR RNA (pre-crRNA), which is further processed by Cas6 in type I and III systems (processing in type I-C CRISPR-Cas systems by Cas5d). In type II CRISPR-Cas systems, crRNA maturation requires tracrRNA, RNase III and Cas9, whereas in type V-A systems Cpf1 alone is sufficient for crRNA maturation. In the interference state of type I systems, Cascade is guided by crRNA to bind the foreign DNA in a sequence-specific manner and subsequently recruits Cas3 that degrades the displaced strand through its 3′–5′ exonucleolytic activity. Type III-A and type III-B CRISPR-Cas systems employ Csm and Cmr complexes, respectively, for cleavage of DNA (red triangles) and its transcripts (black triangles). A ribonucleoprotein complex consisting of Cas9 and a tracrRNA : crRNA duplex targets and cleaves invading DNA in type II CRISPR-Cas systems. The crRNA-guided effector protein Cpf1 is responsible for target degradation in type V systems. Red triangles represent the cleavage sites of the interference machinery.
Figure 2.
Figure 2.
Applications of the CRISPR-Cas9 technology. (a) Cas9 is guided by a sgRNA to induce a double-strand DNA break at a desired genomic locus. The DNA damage can be repaired by NHEJ yielding short random insertions or deletions at the target site. Alternatively, a DNA sequence that shows partial complementarity to the target site can be inserted during HDR for precise genome editing purposes. (b) Mutations in the catalytical domains of Cas9 yield a dead variant (dCas9) that binds but does not cleave DNA. The approach with dCas9 is used for transcriptional repression by binding to the promoter region of a gene and thus blocking the access for the RNA polymerase. Similarly, dCas9 can be fused to a transcriptional repressor. Red crosses represent inhibition of transcription. (c) The fusion of dCas9 to a transcriptional activator stimulates transcription of an adjacent gene by recruiting the RNA polymerase.

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