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  • Review Article
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Beyond editing: repurposing CRISPR–Cas9 for precision genome regulation and interrogation

Nature Reviews Molecular Cell Biology volume 17, pages 5–15 (2016)Cite this article

Subjects

Key Points

  • Beyond gene editing, the CRISPR–Cas9 technology offers a versatile sequence-specific gene regulation 'toolset', by utilizing the nuclease-deficient dCas9, which was designed not to cleave DNA but to precisely and specifically bind DNA when guided by a single guide RNA (sgRNA).

  • In CRISPR interference (CRISPRi), dCas9 is targeted to block transcription and thereby silence genes. Improvements include the fusion of dCas9 to transcriptional repressors for increased repression efficiency.

  • CRISPR activation (CRISPRa) uses dCas9 fusion proteins to recruit transcription activators for targeted gene activation. The use of enhanced dCas9 activation systems allows recruitment of multiple activators with one sgRNA.

  • dCas9 can direct epigenetic modifications at specific genomic locations, through the use of dCas9 fusion proteins that recruit epigenetic modifiers that can alter epigenetic marks at enhancers and other regulatory elements.

  • Rational engineering of the sgRNA molecule by the addition of aptamers allows for the recruitment of transcription repressors or activators, and the simultaneous activation of one gene target and repression of another gene target, using one dCas9 protein.

  • CRISPRi and CRISPRa are highly gene-specific.

  • Applications of dCas9 currently include genome-wide loss-of-function or gain-of function screens, inducible and reversible gene regulation and cell fate engineering.

Abstract

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.

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Figure 1: Gene editing versus gene regulation using Streptococcus pyogenes Cas9 and dCas9.
Figure 2: CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) for transcription repression and activation.
Figure 3: Simultaneous transcription activation and repression.
Figure 4: Applications of the CRISPR–dCas9 technology.

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Acknowledgements

The authors thank the members of the Qi lab for advice and helpful discussions. L.S.Q. acknowledges support from the U.S. National Institutes of Health (NIH) Office of the Director (OD) and National Institute of Dental & Craniofacial Research (NIDCR). A.A.D. acknowledges support through the Milton Safenowitz Post Doctoral Fellowship for ALS Research. This work was supported by NIH R01 DA036858 (to W.A.L. and L.S.Q.), the Howard Hughes Medical Institute (grant to W.A.L.) and NIH DP5 OD017887 (to A.A.D. and L.S.Q.).

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Authors and Affiliations

  1. Department of Bioengineering, Stanford University, Stanford, 94305, California, USA

    Antonia A. Dominguez & Lei S. Qi

  2. Department of Chemical and Systems Biology. Stanford University, Stanford, 94305, California, USA

    Antonia A. Dominguez & Lei S. Qi

  3. Stanford ChEM-H, Stanford University, Stanford, 94305, California, USA

    Antonia A. Dominguez & Lei S. Qi

  4. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, 94158, California, USA

    Wendell A. Lim

  5. Howard Hughes Medical Institute, University of California, San Francisco, 94158, California, USA

    Wendell A. Lim

  6. UCSF Center for Systems and Synthetic Biology, University of California, San Francisco, 94158, California, USA

    Wendell A. Lim

  7. California Institute for Quantitative Biomedical Research (QB3), University of California, San Francisco, 94158, California, USA

    Wendell A. Lim

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PowerPoint slides

Glossary

CRISPR–Cas

(Clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins). CRISPR are bacterial DNA loci containing short repeat segments that match foreign DNA elements. Together with Cas proteins, they form an adaptive immune system in bacteria and archaea, which can acquire sequence segments from foreign DNA and use these sequences to recognize and destroy the foreign target DNA.

Type II CRISPR system

CRISPR–Cas system that encodes cas9, cas1 and cas2 within the CRISPR–cas loci, in addition to a tracrRNA, which is partially complementary to repeats in the CRISPR array.

Single guide RNA

(sgRNA). A synthetic RNA chimera containing a hairpin that links the transactivating CRISPR RNA (tracrRNA) to the crRNA and functions similarly to the native crRNA–tracrRNA duplex, directing Cas9 to a specific genomic locus.

Protospacer-adjacent motif

(PAM). A short sequence in the target DNA (not in the guide RNA) that is necessary for the successful targeting of Cas9. The PAM sequence varies between bacterial species. In Streptococcus pyogenes, it is NGG (which is more effective) or NAG (less effective).

Transactivating CRISPR RNA

(tracrRNA). A small RNA encoded upstream of the CRISPR locus in type II CRISPR systems, with a 24-nucleotide sequence complementary to repeats of the CRISPR RNA (crRNA) precursor transcripts. Essential for the processing of pre-crRNA to mature crRNA.

CRISPR RNA

(crRNA). Small RNAs transcribed from the protospacer within the CRISPR array. Together with the transactivating crRNA (tracrRNA), crRNA guides Cas9 to a specific genomic locus.

Krüppel-associated box

(KRAB). A conserved domain of a transcription repressor that can be fused to DNA-binding proteins for targeted transcription repression.

mSin3 interaction domains

Interaction domains that are present on multiple transcriptional repressor proteins.

VP64

A transcription activator composed of four tandem copies of the herpes simplex virus VP16 activation domain connected by Gly-Ser linkers. VP64 is often fused to DNA-binding proteins for targeted transcription activation.

p65 activation domain

(p65AD). The principal transactivation domain of the 65 kDa polypeptide of the nuclear form of the NF-κB transcription factor.

Single-chain variable fragment

(scFV). A fusion protein in which the epitope-binding regions of the heavy and light chains of an antibody are connected by a short linker peptide and are expressed in soluble form in cells.

Mediator complex

A multi-subunit complex that is required for the transcription of most RNA polymerase II transcripts.

RNA aptamers

RNA molecules that have high affinity and specificity for target molecules.

Optogenetics

A technique that utilizes optics for achieving spatiotemporal gene regulation of cells in living tissues.

CRY–CIB heterodimerizing domains

A light-inducible protein interaction between the blue light-sensitive cryptochrome 2 protein (CRY2) and its interacting partner CIB1 from Arabidopsis thaliana.

Direct lineage reprogramming

The conversion of fully differentiated cells of a certain type into another cell type, while bypassing the intermediate pluripotent state.

Adeno-associated virus (AAV) vectors

Viral vectors with small packaging capacity, commonly used in gene therapy, which can infect both dividing and non-dividing cells and do not integrate into the host genome. AAV vectors have been approved for clinical use.

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Dominguez, A., Lim, W. & Qi, L. Beyond editing: repurposing CRISPR–Cas9 for precision genome regulation and interrogation. Nat Rev Mol Cell Biol 17, 5–15 (2016). https://doi.org/10.1038/nrm.2015.2

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