Genome editing, which involves the precise manipulation of cellular DNA sequences to alter cell fates and organism traits, has the potential to both improve our understanding of human genetics and cure genetic disease. Here I discuss the scientific, technical and ethical aspects of using CRISPR (clustered regularly interspaced short palindromic repeats) technology for therapeutic applications in humans, focusing on specific examples that highlight both opportunities and challenges. Genome editing is—or will soon be—in the clinic for several diseases, with more applications under development. The rapid pace of the field demands active efforts to ensure that this breakthrough technology is used responsibly to treat, cure and prevent genetic disease.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012). This study demonstrates dual RNA-programmed DNA cutting by CRISPR–Cas9 and establishes a sgRNA format to direct Cas9 applications, providing a road map for genome editing in human, animal and plant cells.
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013).
Cho, S. W., Kim, S., Kim, J. M. & Kim, J.-S. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 230–232 (2013).
Knott, G. J. & Doudna, J. A. CRISPR–Cas guides the future of genetic engineering. Science 361, 866–869 (2018).
Hidalgo-Cantabrana, C., Goh, Y. J. & Barrangou, R. Characterization and repurposing of type I and type II CRISPR–Cas systems in bacteria. J. Mol. Biol. 431, 21–33 (2019).
Bao, A. et al. The CRISPR/Cas9 system and its applications in crop genome editing. Crit. Rev. Biotechnol. 39, 321–336 (2019).
Terns, M. P. CRISPR-based technologies: impact of RNA-targeting systems. Mol. Cell 72, 404–412 (2018).
High, K. A. & Roncarolo, M. G. Gene therapy. N. Engl. J. Med. 381, 455–464 (2019).
Pauling, L. et al. Sickle cell anemia, a molecular disease. Science 110, 543–548 (1949).
Ingram, V. M. Gene mutations in human haemoglobin: the chemical difference between normal and sickle cell haemoglobin. Nature 180, 326–328 (1957).
Shieh, P. B. Emerging strategies in the treatment of Duchenne muscular dystrophy. Neurotherapeutics 15, 840–848 (2018).
Min, Y.-L., Bassel-Duby, R. & Olson, E. N. CRISPR correction of Duchenne muscular dystrophy. Annu. Rev. Med. 70, 239–255 (2019).
Long, C. et al. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science 345, 1184–1188 (2014).
Miller, J. C. et al. A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol. 29, 143–148 (2011).
Hoban, M. D. et al. Zinc finger nucleases targeting the β-globin locus drive efficient correction of the sickle mutation in CD34+ cells. Blood 122, 2904 (2013).
Chang, K.-H. et al. Long-term engraftment and fetal globin induction upon BCL11A gene editing in bone-marrow-derived CD34+ hematopoietic stem and progenitor cells. Mol. Ther. Methods Clin. Dev. 4, 137–148 (2017).
Qasim, W. et al. Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells. Sci. Transl. Med. 9, eaaj2013 (2017).
Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl Acad. Sci. USA 109, E2579–E2586 (2012).
Szostak, J. W., Orr-Weaver, T. L., Rothstein, R. J. & Stahl, F. W. The double-strand-break repair model for recombination. Cell 33, 25–35 (1983). The authors proposed a surprising but ultimately correct cellular DNA repair mechanism in which double-stranded breaks are enlarged to double-stranded gaps to initiate genetic recombination, forming the basis for genome editing mediated by DNA repair.
Stark, J. M., Pierce, A. J., Oh, J., Pastink, A. & Jasin, M. Genetic steps of mammalian homologous repair with distinct mutagenic consequences. Mol. Cell. Biol. 24, 9305–9316 (2004). Double-stranded DNA breaks in mammalian cells trigger DNA repair that can introduce site-specific changes in the genome sequence.
Liu, J.-J. et al. CasX enzymes comprise a distinct family of RNA-guided genome editors. Nature 566, 218–223 (2019).
Yan, W. X. et al. Cas13d is a compact RNA-targeting type VI CRISPR effector positively modulated by a WYL-domain-containing accessory protein. Mol. Cell 70, 327–339 (2018).
Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016). A DNA-nicking version of CRISPR–Cas9 was fused to a DNA-editing enzyme that enables targeted nucleotide changes to be introduced at Cas9-directed genome locations.
Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, aaf8729 (2016). CRISPR–Cas9 was fused to a DNA-editing enzyme that enables targeted nucleotide editing at genome locations recognized by Cas9, while avoiding double-stranded DNA breaks.
Sharon, E. et al. Functional genetic variants revealed by massively parallel precise genome editing. Cell 175, 544–557 (2018). This study showed that an RNA template can be used together with a Cas9–reverse transcriptase fusion protein to introduce small targeted changes in cellular genomes without involving double-stranded DNA break repair.
Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019). A CRISPR–Cas9-reverse transcriptase fusion protein was used together with extended guide-RNA templates to introduce small sequence changes within approximately 50 base pairs of the location of Cas9 binding.
Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013). This study demonstrated the use of a catalytically deactivated form of CRISPR–Cas9 for transcriptional control in cells.
Gilbert, L. A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).
Pickar-Oliver, A. & Gersbach, C. A. The next generation of CRISPR–Cas technologies and applications. Nat. Rev. Mol. Cell Biol. 20, 490–507 (2019).
Xu, X. & Qi, L. S. A CRISPR–dCas toolbox for genetic engineering and synthetic biology. J. Mol. Biol. 431, 34–47 (2019).
Grünewald, J. et al. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature 569, 433–437 (2019).
Zhou, C. et al. Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis. Nature 571, 275–278 (2019).
Antoniani, C. et al. Induction of fetal hemoglobin synthesis by CRISPR/Cas9-mediated editing of the human β-globin locus. Blood 131, 1960–1973 (2018).
Chung, J. E. et al. CRISPR–Cas9 interrogation of a putative fetal globin repressor in human erythroid cells. PLoS ONE 14, e0208237 (2019).
Bjurström, C. F. et al. Reactivating fetal hemoglobin expression in human adult erythroblasts through BCL11A knockdown using targeted endonucleases. Mol. Ther. Nucleic Acids 5, e351 (2016).
Liu, N. et al. Direct promoter repression by BCL11A controls the fetal to adult hemoglobin switch. Cell 173, 430–442 (2018).
Shariati, L. et al. Genetic disruption of the KLF1 gene to overexpress the γ-globin gene using the CRISPR/Cas9 system. J. Gene Med. 18, 294–301 (2016).
Martyn, G. E. et al. Natural regulatory mutations elevate the fetal globin gene via disruption of BCL11A or ZBTB7A binding. Nat. Genet. 50, 498–503 (2018).
Grevet, J. D. et al. Domain-focused CRISPR screen identifies HRI as a fetal hemoglobin regulator in human erythroid cells. Science 361, 285–290 (2018).
Martyn, G. E. et al. A natural regulatory mutation in the proximal promoter elevates fetal globin expression by creating a de novo GATA1 site. Blood 133, 852–856 (2019).
Lomova, A. et al. Improving gene editing outcomes in human hematopoietic stem and progenitor cells by temporal control of DNA repair. Stem Cells 37, 284–294 (2019).
Schumann, K. et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc. Natl Acad. Sci. USA 112, 10437–10442 (2015).
Dever, D. P. et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature 539, 384–389 (2016).
DeWitt, M. A. et al. Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells. Sci. Transl. Med. 8, 360ra134 (2016).
Wu, Y. et al. Highly efficient therapeutic gene editing of human hematopoietic stem cells. Nat. Med. 25, 776–783 (2019).
Amoasii, L. et al. Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy. Sci. Transl. Med. 9, eaan8081 (2017).
Gaudelli, N. M. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).
Amoasii, L. et al. Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy. Science 362, 86–91 (2018). This article presents evidence that CRISPR–Cas9 can induce corrective genome edits in sufficient cell numbers in vivo to provide therapeutic benefit in a dog model of DMD.
Zuo, E. et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364, 289–292 (2019).
Sharma, R. et al. In vivo genome editing of the albumin locus as a platform for protein replacement therapy. Blood 126, 1777–1784 (2015). A potential therapeutic strategy in which blood cells are edited to enable the high-level expression of a desired protein is described.
Laoharawee, K. et al. Dose-dependent prevention of metabolic and neurologic disease in murine MPS II by ZFN-mediated in vivo genome editing. Mol. Ther. 26, 1127–1136 (2018).
Maeder, M. L. et al. Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10. Nat. Med. 25, 229–233 (2019). This study explored a method for treating inherited retinal disease using a genome-editing approach that uses an AAV5 vector to deliver the S. aureus Cas9 and sgRNAs to photoreceptor cells by subretinal injection.
Finn, J. D. et al. A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing. Cell Rep. 22, 2227–2235 (2018). Lipid nanoparticle-based delivery of mRNA-encoded Cas9 and sgRNAs provided therapeutically relevant levels of genome editing in the liver in mice.
Hultquist, J. F. et al. A Cas9 ribonucleoprotein platform for functional genetic studies of HIV–host interactions in primary human T cells. Cell Rep. 17, 1438–1452 (2016).
Wang, J.-Z., Wu, P., Shi, Z.-M., Xu, Y.-L. & Liu, Z.-J. The AAV-mediated and RNA-guided CRISPR/Cas9 system for gene therapy of DMD and BMD. Brain Dev. 39, 547–556 (2017).
Mendell, J. R. et al. Single-dose gene-replacement therapy for spinal muscular atrophy. N. Engl. J. Med. 377, 1713–1722 (2017).
Yang, Y. et al. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. Nat. Biotechnol. 34, 334–338 (2016).
Lau, C.-H. & Suh, Y. In vivo genome editing in animals using AAV–CRISPR system: applications to translational research of human disease. F1000Res. 6, 2153 (2017).
Bengtsson, N. E. et al. Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy. Nat. Commun. 8, 14454 (2017).
Nelson, C. E. et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science 351, 403–407 (2016). In this study, the feasibility of achieving therapeutically meaningful levels of genome editing in affected tissues in a mouse model of muscular dystrophy was demonstrated.
Tabebordbar, M. et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science 351, 407–411 (2016). The feasibility of achieving therapeutically meaningful levels of genome editing in affected tissues in a mouse model of muscular dystrophy was demonstrated.
Li, H. et al. Inhibition of HBV expression in HBV transgenic mice using AAV-delivered CRISPR–SaCas9. Front. Immunol. 9, 2080 (2018).
Kim, E. et al. In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nat. Commun. 8, 14500 (2017).
Charlesworth, C. T. et al. Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nat. Med. 25, 249–254 (2019).
Simhadri, V. L. et al. Prevalence of pre-existing antibodies to CRISPR-associated nuclease Cas9 in the USA population. Mol. Ther. Methods Clin. Dev. 10, 105–112 (2018).
Sandoval, I. M., Kuhn, N. M. & Manfredsson, F. P. Multimodal production of adeno-associated virus. Methods Mol. Biol. 1937, 101–124 (2019).
Sandro, Q., Relizani, K. & Benchaouir, R. AAV production using baculovirus expression vector system. Methods Mol. Biol. 1937, 91–99 (2019).
Strobel, B. et al. Standardized, scalable, and timely flexible adeno-associated virus vector production using frozen high-density HEK-293 cell stocks and CELLdiscs. Hum. Gene Ther. Methods 30, 23–33 (2019).
Miller, J. B. et al. Non-viral CRISPR/Cas gene editing in vitro and in vivo enabled by synthetic nanoparticle co-delivery of Cas9 mRNA and sgRNA. Angew. Chem. Int. Edn 56, 1059–1063 (2017).
Wang, M. et al. Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles. Proc. Natl Acad. Sci. USA 113, 2868–2873 (2016).
Yeh, W.-H., Chiang, H., Rees, H. A., Edge, A. S. B. & Liu, D. R. In vivo base editing of post-mitotic sensory cells. Nat. Commun. 9, 2184 (2018).
Gao, X. et al. Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents. Nature 553, 217–221 (2018).
Zuris, J. A. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73–80 (2015).
Staahl, B. T. et al. Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes. Nat. Biotechnol. 35, 431–434 (2017).
Ding, Y. et al. Gold nanoparticles for nucleic acid delivery. Mol. Ther. 22, 1075–1083 (2014).
Glass, Z., Li, Y. & Xu, Q. Nanoparticles for CRISPR–Cas9 delivery. Nat. Biomed. Eng. 1, 854–855 (2017).
Lee, K. et al. Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair. Nat. Biomed. Eng. 1, 889–901 (2017).
Sago, C. D. et al. High-throughput in vivo screen of functional mRNA delivery identifies nanoparticles for endothelial cell gene editing. Proc. Natl Acad. Sci. USA 115, E9944–E9952 (2018).
Qin, W. & Wang, H. Delivery of CRISPR–Cas9 into mouse zygotes by electroporation. Methods Mol. Biol. 1874, 179–190 (2019).
Tanihara, F. et al. Generation of a TP53-modified porcine cancer model by CRISPR/Cas9-mediated gene modification in porcine zygotes via electroporation. PLoS ONE 13, e0206360 (2018).
Xu, L. et al. CRISPR-mediated genome editing restores dystrophin expression and function in mdx mice. Mol. Ther. 24, 564–569 (2016).
Kim, S., Kim, D., Cho, S. W., Kim, J. & Kim, J.-S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014). This study demonstrated the use of purified protein–guide RNA complexes for genome editing in human cells.
Lin, S., Staahl, B. T., Alla, R. K. & Doudna, J. A. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 3, e04766 (2014).
Gaj, T. et al. Targeted gene knock-in by homology-directed genome editing using Cas9 ribonucleoprotein and AAV donor delivery. Nucleic Acids Res. 45, e98 (2017).
Rouet, R. et al. Receptor-mediated delivery of CRISPR–Cas9 endonuclease for cell-type-specific gene editing. J. Am. Chem. Soc. 140, 6596–6603 (2018).
Yin, J. et al. Potent protein delivery into mammalian cells via a supercharged polypeptide. J. Am. Chem. Soc. 140, 17234–17240 (2018).
Riley, R. S., June, C. H., Langer, R. & Mitchell, M. J. Delivery technologies for cancer immunotherapy. Nat. Rev. Drug Discov. 18, 175–196 (2019).
Sun, Y. et al. Advances of blood cell-based drug delivery systems. Eur. J. Pharm. Sci. 96, 115–128 (2017).
Sharma, P. et al. Efficient intracellular delivery of biomacromolecules employing clusters of zinc oxide nanowires. Nanoscale 9, 15371–15378 (2017).
Kim, D., Luk, K., Wolfe, S. A. & Kim, J.-S. Evaluating and enhancing target specificity of gene-editing nucleases and deaminases. Annu. Rev. Biochem. 88, 191–220 (2019).
Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nat. Biotechnol. 36, 765–771 (2018).
Poirot, L. et al. Multiplex genome-edited T-cell manufacturing platform for “off-the-shelf” adoptive T-cell immunotherapies. Cancer Res. 75, 3853–3864 (2015).
Bak, R. O. et al. Multiplexed genetic engineering of human hematopoietic stem and progenitor cells using CRISPR/Cas9 and AAV6. eLife 6, e27873 (2017).
Tichy, E. D. et al. Mouse embryonic stem cells, but not somatic cells, predominantly use homologous recombination to repair double-strand DNA breaks. Stem Cells Dev. 19, 1699–1711 (2010).
Buechele, C. et al. MLL leukemia induction by genome editing of human CD34+ hematopoietic cells. Blood 126, 1683–1694 (2015).
Maddalo, D. et al. In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system. Nature 516, 423–427 (2014).
NIST. NIST Genome Editing Consortium https://www.nist.gov/programs-projects/nist-genome-editing-consortium (NIST, 2017).
Gilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).
Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247 (2016). CRISPR–Cas9 fusion proteins were used to introduce targeted epigenetic changes into cellular genomes.
Wilson, R. C. & Carroll, D. The daunting economics of therapeutic genome editing. CRISPR J. 2, 280–284 (2019).
Wagner, D. L. et al. High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population. Nat. Med. 25, 242–248 (2019).
Haapaniemi, E., Botla, S., Persson, J., Schmierer, B. & Taipale, J. CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response. Nat. Med. 24, 927–930 (2018).
Ihry, R. J. et al. p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells. Nat. Med. 24, 939–946 (2018).
Schiroli, G. et al. Precise gene editing preserves hematopoietic stem cell function following transient p53-mediated DNA damage response. Cell Stem Cell 24, 551–565.e8 (2019).
Xu, L. et al. CRISPR-edited stem cells in a patient with HIV and acute lymphocytic leukemia. N. Engl. J. Med. 381, 1240–1247 (2019).
Porteus, M. H. A new class of medicines through DNA editing. N. Engl. J. Med. 380, 947–959 (2019).
Liang, P. et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell 6, 363–372 (2015).
Fogarty, N. M. E. et al. Genome editing reveals a role for OCT4 in human embryogenesis. Nature 550, 67–73 (2017).
Tang, L. et al. CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein. Mol. Genet. Genomics 292, 525–533 (2017).
Ma, H. et al. Correction of a pathogenic gene mutation in human embryos. Nature 548, 413–419 (2017).
Kaul, S., Heitner, S. B. & Mitalipov, S. Sarcomere gene mutation correction. Eur. Heart J. 39, 1506–1507 (2018).
Egli, D. et al. Inter-homologue repair in fertilized human eggs? Nature 560, E5–E7 (2018).
Ormond, K. E. et al. Human germline genome editing. Am. J. Hum. Genet. 101, 167–176 (2017).
J.A.D. thanks M. Hochstrasser and M. Triplet for extensive expert assistance with manuscript editing, formatting, referencing and illustrations. D. Carroll, F. Urnov and R. Wilson provided comments on the manuscript. T. Tolpa created the artwork with input from M. Hochstrasser and E. Stahl, and with support from the Innovative Genomics Institute. J.A.D. is an investigator of the Howard Hughes Medical Institute (HHMI) and a Paul Allen Distinguished Investigator. In addition to funding from HHMI and the Paul Allen Frontiers Group, research in the Doudna laboratory is supported by the Defense Advanced Research Projects Agency (DARPA) (award HR0011-17-2-0043), the William M. Keck Foundation, a Collaborative MS Research Center Award from the National Multiple Sclerosis Society, the Centers for Excellence in Genomic Science of the National Institutes of Health under award number RM1HG009490, the Somatic Cell Genome Editing Program of the Common Fund of the National Institutes of Health under award number U01AI142817-02 and the National Science Foundation under award number 1817593.
J.A.D. is a co-founder of Caribou Biosciences, Editas Medicine, Intellia Therapeutics, Scribe Therapeutics and Mammoth Biosciences; a scientific adviser to Caribou Biosciences, Intellia Therapeutics, Scribe Therapeutics, Synthego, Inari and eFFECTOR Therapeutics; and a director of Johnson & Johnson. The Regents of the University of California have patents issued and pending for CRISPR-related technologies on which J.A.D. is an inventor.
Peer review information Nature thanks R. Alta Charo and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Doudna, J.A. The promise and challenge of therapeutic genome editing. Nature 578, 229–236 (2020). https://doi.org/10.1038/s41586-020-1978-5
Genome Medicine (2021)
Stem Cell Research & Therapy (2021)
Nature Communications (2021)
Nature Communications (2021)
Nature Protocols (2021)