CRISPR–Cas tools are easily programmable RNA-guided nucleases that are derived from microbial adaptive immune systems and enable rapid genome engineering in vitro and in vivo.
Paired with the rapid expansion of genomic information, CRISPR–Cas enables facile genetic manipulation, even in previously difficult contexts such as human cells. Gene knockout via error-prone repair, such as non-homologous end joining (NHEJ), works well in nearly all cell types, whereas knock-in via homology-directed repair (HDR) is more variable.
CRISPR–Cas holds the promise to transform the discovery and development of therapies to treat complex heritable and somatic disorders. Early applications in the field of cancer immunotherapy are entering clinical trials.
CRISPR–Cas gene editing expedites the generation of accurate cellular and animal models of human disease to facilitate drug discovery and validation. CRISPR–Cas could be used in all species that are commonly used during a typical preclinical drug development campaign.
The low barrier to deploying CRISPR–Cas technology has enabled its rapid spread throughout the scientific community and is revolutionizing biomedical research. CRISPR–Cas systems are excellent tools for large-scale functional screens using gene knockout (CRISPRn), inhibition (CRISPRi) or activation (CRISPRa).
Further evolution of CRISPR–Cas9 may enable cures for Mendelian diseases in somatic tissues by directly correcting the underlying disease-causing mutations. Pioneering work with zinc-finger nuclease (ZFN)-based and transcription activator-like effector nuclease (TALEN)-based therapies will determine the path to therapeutic gene editing with CRISPR–Cas.
The recent development of CRISPR–Cas systems as easily accessible and programmable tools for genome editing and regulation is spurring a revolution in biology. Paired with the rapid expansion of reference and personalized genomic sequence information, technologies based on CRISPR–Cas are enabling nearly unlimited genetic manipulation, even in previously difficult contexts, including human cells. Although much attention has focused on the potential of CRISPR–Cas to cure Mendelian diseases, the technology also holds promise to transform the development of therapies to treat complex heritable and somatic disorders. In this Review, we discuss how CRISPR–Cas can affect the next generation of drugs by accelerating the identification and validation of high-value targets, uncovering high-confidence biomarkers and developing differentiated breakthrough therapies. We focus on the promises, pitfalls and hurdles of this revolutionary gene-editing technology, discuss key aspects of different CRISPR–Cas screening platforms and offer our perspectives on the best practices in genome engineering.
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The authors thank members of the Doudna and Corn laboratories, as well as F. Urnov for insightful comments and discussions. C.F. is supported by a US National Institutes of Health K99/R00 Pathway to Independence Award (K99GM118909) from the National Institute of General Medical Sciences (NIGMS). The Innovative Genomics Initiative (IGI) is supported by the Li Ka Shing Foundation.
J.A.D. is employed by the Howard Hughes Medical Institute (HHMI) and works at the University at California (UC), Berkeley, USA. UC Berkeley and HHMI have patents pending for CRISPR technologies on which J.A.D. and J.E.C. are inventors. J.A.D. is the executive director and J.E.C is the scientific director of the Innovative Genomics Initiative (IGI) at UC Berkeley and University of California, San Francisco. J.A.D. is a co-founder of Editas Medicine, Intellia Therapeutics and Caribou Biosciences, and a scientific adviser to Caribou, Intellia, eFFECTOR Therapeutics and Driver. J.E.C. is a consultant to or has funded research collaborations with AstraZeneca, CRISPR Therapeutics, Editas Medicine, Genentech, Intellia and Pfizer.
- Non-homologous end joining
(NHEJ). The repair of double-strand DNA breaks by direct ligation of the broken ends. No homology is required to promote the end-joining reaction, and it can result in the introduction of small non-templated insertions or deletions (indels).
- Homology-directed repair
(HDR). The repair of double-strand DNA breaks using an endogenous or exogenous DNA template with homology to regions flanking the break.
The activation of transcription through RNA-guided recruitment of a catalytically inactive Cas9 fused to transcriptional activators.
The inhibition of transcription through RNA-guided recruitment of a catalytically inactive Cas9 fused to transcriptional repressors.
- CRISPR nuclease
(CRISPRn). Targeting a DNA sequence with catalytically active Cas9 to generate a double-strand break or a nick.
The process of breeding a hybrid organism with an individual genetically similar to one of its parents, with the objective of diluting the genetic contribution of the other parent to subsequent generations.
- Protospacer adjacent motif
(PAM). Short genomic sequence adjacent to the sequence targeted by the guide RNA that is required for recognition by Cas effectors. This sequence varies based on the identity of the effector (for example, Cas9 versus Cpf1) and species (for example, Streptococcus pyogenes versus Francisella novicida).
- Investigational new drugs
(INDs). A designation used to describe drugs that have permission from the US Food and Drug Administration (FDA) to be shipped across state lines, thus allowing these drugs to be tested in human clinical trials. IND applications are reviewed by the FDA to ensure that testing of the drug in humans does not pose excessive risk to the patient.
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Fellmann, C., Gowen, B., Lin, PC. et al. Cornerstones of CRISPR–Cas in drug discovery and therapy. Nat Rev Drug Discov 16, 89–100 (2017). https://doi.org/10.1038/nrd.2016.238
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