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, 17 (1), 5-15

Beyond Editing: Repurposing CRISPR-Cas9 for Precision Genome Regulation and Interrogation


Beyond Editing: Repurposing CRISPR-Cas9 for Precision Genome Regulation and Interrogation

Antonia A Dominguez et al. Nat Rev Mol Cell Biol.


The bacterial CRISPR-Cas9 system has emerged as a multifunctional platform for sequence-specific regulation of gene expression. This Review describes the development of technologies based on nuclease-deactivated Cas9, termed dCas9, for RNA-guided genomic transcription regulation, both by repression through CRISPR interference (CRISPRi) and by activation through CRISPR activation (CRISPRa). We highlight different uses in diverse organisms, including bacterial and eukaryotic cells, and summarize current applications of harnessing CRISPR-dCas9 for multiplexed, inducible gene regulation, genome-wide screens and cell fate engineering. We also provide a perspective on future developments of the technology and its applications in biomedical research and clinical studies.


Figure 1
Figure 1. Gene editing versus gene regulation using Streptococcus pyogenes Cas9 and dCas9
a | The S. pyogenes Cas9 endonuclease consists of a nuclease (NUC) lobe and a recognition (REC) lobe. Cas9 is targeted to specific DNA sequences by direct pairing of the chimeric single guide RNA (sgRNA) with the target DNA. This targeting relies on the presence of a 5′ protospacer-adjacent motif (PAM) in the DNA, which in S. pyogenes is usually NGG. Binding mediates cleavage of the target sequence by two nuclease domains, RuvC1 and HNH. b | The S. pyogenes dCas9 protein contains mutations in its RuvC1 (D10A) and HNH (H841A) domains, which inactivate its nuclease function (circles). dCas9 retains the ability to target specific sequences through the sgRNA and PAM. dCas9 binding downstream of the transcription start site (TSS) can block transcription elongation by blocking RNA polymerase II (Pol II) or the binding of important transcription factors (Txn).
Figure 2
Figure 2. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) for transcription repression and activation
a | Transcription repression by nuclease-deficient Cas 9 (dCas9) can be improved by fusing dCas9 with different repressor domains (red dashed ovals), including MAX-interacting protein 1 (MXI1), Krüppel-associated box (KRAB) domain or four concatenated mSin3 domains (SID4X), to either amino or carboxyl termini. b | Initial strategies for transcription activation included fusing dCas9 with different activation domains (green dashed ovals), including multiple repeats of the herpes simplex VP16 activation domain (VP64 or VP160) or the nuclear factor-κB (NF-κB) transactivating subunit activation domain (p65AD). Improved levels of transcription activation were achieved with VP64 fused at both N and C termini. These methods require the use of multiple single guide RNAs (sgRNAs; different shades of orange) to recruit multiple dCas9 fusion proteins to achieve efficient transcription activation. c | In methods for enhanced transcription activation, it is sufficient to use only one sgRNA to recruit one dCas9 per target gene. The SunTag activation method uses an array of small peptide epitopes (blue circles) fused to the C terminus of dCas9 to recruit multiple copies of single-chain variable fragment (scFV) fused to super folder GFP (sfGFP; for improving protein folding), fused to VP64. The synergistic tripartite activation method (VPR) uses a tandem fusion of three transcription activators, VP64, p65 and the Epstein–Barr virus R transactivator (Rta), to achieve enhanced transcription activation. d | The aptamer-based recruitment system (synergistic activation mediator (SAM)) utilizes dCas9 with a sgRNA encoding MS2 RNA aptamers at the tetraloop and the second stem–loop (shown in dark green) to recruit the MS2 coat protein (MCP) that is fused to two activators, p65 and heat shock factor 1 (HSF1). Additionally, VP64 is fused to dCas9. e | Epigenetic regulation can be carried out by fusion of epigenetic regulators to dCas9. Fusion of the histone demethylase LSD1 to Neisseria meningitidis Cas9 removes the histone 3 Lys4 dimethylation (H3K4me2) mark from targeted distal enhancers, leading to transcription repression. f | The fusion of the catalytic core of the histone acetyltransferase p300 (p300Core) to dCas9 can acetylate H3K27 (H3K27ac) at targeted proximal and distal enhancers, which leads to transcription activation.
Figure 3
Figure 3. Simultaneous transcription activation and repression
Multiplexed transcription activation and repression is carried out using single guide RNAs (sgRNAs) modified with RNA aptamers, termed scaffold RNAs (scRNAs). The com aptamer recruits the aptamer-binding protein, Com, which was fused to a Krüppel-associated box (KRAB) domain for transcription repression of β-1,4-N-acetyl-galactosaminyl transferase 1 (B4GALNT1). In the same cells, two MS2 aptamers were used to recruit the MS2 coat protein (MCP) fused to VP64 for transcription activation of C-X-C chemokine receptor type 4 (CXCR4).
Figure 4
Figure 4. Applications of the CRISPR–dCas9 technology
a | Overview of pooled screening using CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa). Growth-based screens identify targeting single guide RNAs (sgRNAs) that confer growth advantage or disadvantage on the basis of sgRNA enrichment or depletion in the final population, assessed using deep sequencing. b | Optogenetics-based transcription control using light-dependent peptide heterodimerization. The amino-terminal fragment of CIB1 (CIBN) is fused to both the amino and carboxyl termini of dCas9. The blue light-sensitive cryptochrome 2 protein CRY undergoes a conformational change in the presence of blue light that enables its heterodimerization with CIBN, thus recruiting activation domains (green dashed oval) such as the herpes simplex virus VP16 activation domain (VP64) or the nuclear factor-κB (NF-κB) transactivating subunit activation domain (p65AD). c | In this chemically inducible system for transcription activation, rapamycin induces the dimerization of FK506-binding protein (FKBP) and the FKBP–rapamycin-binding domain (FRB). Thus, a dCas9 protein that is split into dCas9 (N terminus)–FRB and FKBP–dCas9 (C terminus)–VP64 can be reassembled by the introduction of rapamycin. d | The targeted activation of octamer-binding 4 (OCT4) with dCas9–VP192 (12 repeats of VP16) can replace transgenic expression of OCT4 to achieve reprogramming from a differentiated cell to a pluripotent stem cell, but this also requires the transgenic expression of SRY-box 2 (SOX2), Krüppel-like factor 4 (KLF4), LIN-28 homologue A (LIN28) and MYC, and the knockdown (KD) of p53. e | Fusion of two VP64 domains to dCas9 induced the expression of myogenic differentiation 1 (Myod1) in mouse embryonic fibroblasts, which caused direct cell reprogramming into skeletal myocytes. f | Activation of neurogenin 2 (NGN2) and neuronal differentiation 1 (NEUROD1), using dCas9–VPR (where VPR is a complex of VP64, p65AD and Epstein–Barr virus R transactivator Rta) and a mixed pool of sgRNAs, directed the differentiation of pluripotent stem cells into neuron-like cells.

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