The next generation of CRISPR–Cas technologies and applications

Abstract

The prokaryote-derived CRISPR–Cas genome editing systems have transformed our ability to manipulate, detect, image and annotate specific DNA and RNA sequences in living cells of diverse species. The ease of use and robustness of this technology have revolutionized genome editing for research ranging from fundamental science to translational medicine. Initial successes have inspired efforts to discover new systems for targeting and manipulating nucleic acids, including those from Cas9, Cas12, Cascade and Cas13 orthologues. Genome editing by CRISPR–Cas can utilize non-homologous end joining and homology-directed repair for DNA repair, as well as single-base editing enzymes. In addition to targeting DNA, CRISPR–Cas-based RNA-targeting tools are being developed for research, medicine and diagnostics. Nuclease-inactive and RNA-targeting Cas proteins have been fused to a plethora of effector proteins to regulate gene expression, epigenetic modifications and chromatin interactions. Collectively, the new advances are considerably improving our understanding of biological processes and are propelling CRISPR–Cas-based tools towards clinical use in gene and cell therapies.

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Fig. 1: Overview of the main CRISPR–Cas gene editing tools.
Fig. 2: Genome editing strategies.
Fig. 3: RNA-targeting tools and their applications.
Fig. 4: Targeted gene regulation and other applications.

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Acknowledgements

This work was supported by an Allen Distinguished Investigator Award from the Paul G. Allen Frontiers Group, Open Philanthropy, US National Institutes of Health grants R01DA036865, R01AR069085, R21NS103007, R33DA041878, P30AR066527, R41GM119914, R41AI136755, U01HG007900, UM1HG009428 and UG3TR002142 and National Science Foundation grants EFRI-1830957 and DMR-1709527. A.P.-O. is supported by a Pfizer–NCBiotech Distinguished Postdoctoral Fellowship.

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Nature Reviews Molecular Cell Biology thanks J. Chen, R. Platt and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Both authors equally contributed to researching data for the article, the discussion of content, writing of the manuscript and its editing before submission.

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Correspondence to Charles A. Gersbach.

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Competing interests

C.A.G. and A.P.-O. are inventors on patent applications related to CRISPR technologies. C.A.G. is a scientific adviser to Element Genomics, Locus Biosciences and Sarepta Therapeutics.

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Supplementary information

Glossary

Spacer

The interchangeable portion of the guide RNA that is complementary to the targeted sequence.

Directed evolution

Method of generating and selecting for nucleic acid or protein variants with desirable properties.

crRNA arrays

In bacterial genomes, series of spacers flanked by repeats, which are transcribed as a single pre-CRISPR RNA array and subsequently processed into individual CRISPR RNAs. 

Indels

Small insertions or deletions of nucleotides at repair sites of DNA double-strand breaks.

Stress granules

Cytosolic membraneless bodies with high concentrations of RNA and/or proteins that form in different cell stress conditions.

Microsatellite-repeat expansion

Repetitive DNA sequences that can expand between generations and encode RNAs that are toxic to cells and cause neurological disorders.

Anti-CRISPR protein

A protein that interacts with and inhibits CRISPR–Cas activity

DNase I hypersensitive sites

Chromatin regions accessible to the enzyme DNase I; generally denote gene-activity-permissive chromatin.

gRNA scaffolds

The backbone (invariable) portions of guide RNAs, which are recognized by Cas proteins.

gRNA aptamer

RNA structures added to the guide RNA scaffold, which can bind specific effector molecules.

Photobleaching

Reduction in the intensity of fluorescence owing to the imaging of a sample over time.

Chimeric antigen receptors

T cell receptors engineered to recognize a specific antigen.

Universal cell donors

Cells engineered to avoid recognition by a recipient immune system.

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Pickar-Oliver, A., Gersbach, C.A. The next generation of CRISPR–Cas technologies and applications. Nat Rev Mol Cell Biol 20, 490–507 (2019). https://doi.org/10.1038/s41580-019-0131-5

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