Background
Highlights
- CRISPR-Cas9 is a powerful tool to modulate transcription in wide range of cell types.
- An expanding set of CRISPR-based transcription effectors is available.
- Gene networks can be efficiently probed and modified for biotechnology applications.[1]
CRISPR-Cas9 has recently emerged as a promising system for multiplexed genome editing as well as epigenome and transcriptome perturbation. Due to its specificity, ease of use and highly modular programmable nature, it has been widely adopted for a variety of applications such as genome editing, transcriptional inhibition and activation, genetic screening, DNA localization imaging, and many more. In this review, we will discuss non-editing applications of CRISPR-Cas9 for transcriptome perturbation, metabolic engineering, and synthetic biology.[1]
Transcriptional regulation with CRISPR-Cas9
Figure 1. Overview of Cas9 nuclease and dCas9-based transcription factors. (a) Wild-type Cas9 endonuclease guided by crRNA:tracrRNA to a specific site in DNA creates a double-stranded DNA break. (b) dCas9, nuclease deactivated mutant of Cas9, is an RNA programmable DNA binding protein. It can act as a steric repressor of transcription in prokaryotes and eukaryotes. sgRNA is an artificial chimeric molecule consisting of crRNA and tracrRNA molecules connected with a short loop. (c) dCas9 fusion with various transcription effectors can be used to repress or activate transcription. (d) Effector domains can be recruited by sgRNA in addition to dCas9 for enhanced activity. (e) sgRNA can be modified with specific protein binding hairpins to concurrently recruit repressor or activator domains in the same cell.
[1] Crystal structure of a CRISPR RNA-guided surveillance complex, Cascade, bound to a ssDNA target[2]
The . The body is formed by a helical filament of six Cas7 subunits (Cas7.1 to 7.6) wrapped around the crRNA guide, with a head-to-tail dimer of Cse2 (Cse2.1 and Cse2.2) at the belly. Cas6e and the 3′ handle of crRNA cap the Cas7 filament at the head while Cas5 and the 5′ handle cap the tail. The N-terminal base of Cse1 is positioned at the tail of the filament; the C-terminal four-helix bundle contacts Cse2.2. The ssDNA target is juxtaposed to the guide region of the crRNA in a groove formed by the Cas7 filament, the four-helix bundle of Cse1, and the Cse2 dimer.
The do not twist around one another in a helix, but instead adopt an underwound ribbon-like structure reminiscent of a ladder. The 5′ and 3′ ends of the curved target strand are ~102Å apart, roughly the length of straight B-form dsDNA with an identical sequence (~107 Å). The crRNA (green) and ssDNA target (orange) are displayed in a spheres representation. Underwinding is facilitated by (ribbon representation of the crRNA and ssDNA). . Disrupted RNA and DNA nucleotides are colored red and blue, respectively.
Structure of the Cas7 subunit.
Within Cascade, the , with a pitch of ~135Å, around the guide target hybrid. The filament is arranged such that the . .
Stabilization of the guide-target hybrid by Cas7, Cse1, and Cse2.
. 5-bp segment is colored red. Extensive contacts between the guide region of the crRNA and the Cas7 filament bury a large portion of the crRNA backbone, leaving the bases solvent-exposed. The absence of direct contacts between protein side chains and bases of the crRNA explains the lack of sequence specificity by Cascade for the guide sequence. . The DNA target has been removed for clarity. Intercalation by Met166 from Cas7 is highlighted. , while the . Of note, the . . Each displaced RNA nucleotide adopts the syn conformation, is similarly positioned above the backbone of the downstream RNA, and is contacted by . Overview of the . The proteins are represented as rockets, the DNA as a surface. The positions of the disrupted DNA nucleotides (royal blue) are indicated.
Interactions capping the tail of Cascade
. The structure of Cascade reveals that Cas5 is structurally related to Cas7, as it consists of a palm(residues 1 to 78 and 115 to 224) and a thumb (residues 79 to 114) domain, but lacks a fingers domain. . Close-up view of the .
Crystal structure of E. coli Cascade bound to a PAM-containing dsDNA target at 2.45 angstrom resolution[3]
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