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F1000Prime Rep
2013 Nov 01;5:47. doi: 10.12703/P5-47.
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Control of gene expression by CRISPR-Cas systems.
Bikard D
,
Marraffini LA
.
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Clustered regularly interspaced short palindromic repeats (CRISPR) loci and their associated cas (CRISPR-associated) genes provide adaptive immunity against viruses (phages) and other mobile genetic elements in bacteria and archaea. While most of the early work has largely been dominated by examples of CRISPR-Cas systems directing the cleavage of phage or plasmid DNA, recent studies have revealed a more complex landscape where CRISPR-Cas loci might be involved in gene regulation. In this review, we summarize the role of these loci in the regulation of gene expression as well as the recent development of synthetic gene regulation using engineered CRISPR-Cas systems.
Figure 1. Type II CRISPR-Cas systems(a) Genetic organization of the type II-A CRISPR-Cas system of Streptococcus pyogenes SF370. The cas operon is composed of four genes, cas9, cas1, cas2, and csn2; the last three are thought to be involved in the acquisition of new spacer sequences. This operon is followed by the CRISPR array containing seven repeats (white boxes, 36 nt long) and six spacers (numbered, colored boxes, 30 nt long) that match different S. pyogenes bacteriophages. Preceding the cas operon and transcribed in the other direction is the trans-activating CRISPR RNA (tracrRNA) gene, which encodes a small RNA with homology to the repeat sequences. (b) CRISPR RNA (crRNA) processing. The CRISPR locus is transcribed as a long precursor (the crRNA precursor) containing repeats (grey line) and spacers (colored lines). Assisted by Cas9, the tracrRNA interacts with each repeat sequence to generate a double-stranded RNA (dsRNA) that is cleaved by RNase III, thus liberating the small crRNAs from the precursor. Further processing at the 5′ end of the crRNA shortens the length of the guide sequence to 20 nt. Black arrowheads indicate the sites of RNA processing. (c) Targeting. Cas9 scans the genome (dsDNA) of the invader to find a region of complementarity with the guide crRNA and introduces a dsDNA cut using two independent nuclease domains, one for each DNA strand, RuvC and HNH. A requirement for cleavage is the presence of an NGG sequence (known as protospacer adjacent motif, or PAM) immediately downstream of the target site. Abbreviations: cas, CRISPR-associated; CRISPR, clustered regularly interspaced short palindromic repeats; nt, nucleotide.
Figure 2. CRISPR-mediated regulation of gene expression in Francisella novicida(a) Genetic organization of the type II-B CRISPR-Cas system present in F. novicida. It contains 14 different spacer sequences and cas4 instead of csn2. Unique to this system is the presence of a small CRISPR-Cas-associated RNA (scaRNA) transcribed in the same orientation as the CRISPR array. (b) Trans-activating CRISPR RNA (tracrRNA):scaRNA-mediated gene repression. The 5′ end of the tracrRNA is homologous to the repeat sequences (yellow line). This allows its interaction with the scaRNA and its incorporation within Cas9. The 3′ end of the tracrRNA has homology to the bacterial lipoprotein (BLP) messenger RNA and directs the degradation/cleavage of this transcript by an unknown mechanism. Abbreviations: cas, CRISPR-associated; CRISPR, clustered regularly interspaced short palindromic repeats.
Figure 3. RNA-guided gene repression using engineered CRISPR-Cas systems(a) Mutation of the essential residues of the RuvC and HNH nuclease domains converts Cas9 into a CRISPR RNA (crRNA)-guided DNA-binding protein (dCas9). Directing dCas9 to promoter sequences (either the top or bottom DNA strand) results in prevention of transcription initiation, most likely by preventing the binding of RNA polymerase (RNAP) to the promoter elements. (b) Directing dCas9 to the open reading frame regions of genes (with a crRNA that interacts with the coding but not the template strand) results in a transcription stop, presumably because the dCas9 complex blocks RNAP elongation. Arrows indicate the transcription start site. Abbreviations: cas, CRISPR-associated; CRISPR, clustered regularly interspaced short palindromic repeats; tracrRNA, trans-activating CRISPR RNA.
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