Nuclease-mediated genome editing, using TALEN or CRISPR/Cas9, has exploded onto the scene in recent years, with hundreds of research papers and patents being published. These technologies hold great promise for treating a wide range of diseases by repairing defective genes or by inactivating undesirable genes such as viral genes or oncogenes in patients.
Already, CRISPR has been used to restore sight to blind mice1, remove and prevent HIV infection2-5, cure muscular dystrophy in mice6-8, and to treat several other disease animal models. Several companies have already launched efforts to develop genome editing-based therapies for clinical use. However, as always with medical applications, patient safety is vitally important, and there is concern that CRISPR/Cas9 may have adverse effects due to off-target mutations or activation of an immune response.
Now, Schaefer et al. have directly examined the frequency of mutations induced by CRISPR/Cas9 in mice, with shocking results9. The group performed whole-genome sequencing on CRISPR-treated mice at a depth sufficient to accurately identify small insertions and deletions (indels) as well as single-nucleotide variants (SNVs). They found a very large number of SNVs (~1700 per mouse) caused by CRISPR/Cas9 that are not associated with the expected target sites of the CRISPR gRNA. This disagrees with the widely held expectation that CRISPR mostly causes indels, and that mutations at off-target sites are rare and due to sequence similarities of these sites to the gRNA target site. In fact, the majority of the SNVs that Schaefer et al. identified were not predicted by in silico models for off-target CRISPR/Cas9 activity. Furthermore, the widespread and unexplained distribution of SNVs in CRISPR/Cas9-treated mice, including in protein-coding and non-coding RNA genes, emphasizes the importance of further investigating and understanding this phenomenon before genome editing-based treatments find their way to patients.
Although this new result is a serious blow to the momentum of CRISPR/Cas9 towards clinical use, genome-editing technology remains very promising for the future of medicine and research. There is obvious potential for improving CRISPR/Cas9 specificity, as well as for utilizing other genome-editing technologies such as TALEN. In the research arena, other genome modification technologies are available, such as new, rapid, ES cell-based knockout and knockin approaches.
- Wu, W.H. et al. CRISPR repair reveals causative mutation in a preclinical model of retinitis pigmentosa. Mol Ther. 2016;24:1388–1394.
- Ebina H, Misawa N, Kanemura Y, Koyanagi Y. Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Scientific Reports. 2013;3:2510.
- Hu W, Kaminski R, Yang F, Zhang Y, Cosentino L, Li F, Luo B, Alvarez-Carbonell D, Garcia-Mesa Y, Karn J, Mo X, Khalili K. RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection. PNAS. 2014;111:11461-6.
- Mandal PK, Ferreira LM, Collins R, Meissner TB, Boutwell CL, Friesen M, Vrbanac V, Garrison BS, Stortchevoi A, Bryder D, Musunuru K, Brand H, Tager AM, Allen TM, Talkowski ME, Rossi DJ, Cowan CA. Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9. Cell Stem Cell. 2014;15:643-52.
- Liao HK, Gu Y, Diaz A, Marlett J, Takahashi Y, Li M, Suzuki K, Xu R, Hishida T, Chang CJ, Esteban CR, Young J, Izpisua Belmonte JC. Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells. Nature Communications. 2015;6:6413.
- Tabebordbar M, Zhu K, Cheng JK, Chew WL, Widrick JJ, Yan WX, Maesner C, Wu EY, Xiao R, Ran FA, Cong L, Zhang F, Vandenberghe LH, Church GM, Wagers AJ. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science. 2016;351:407-11.
- Nelson CE, Hakim CH, Ousterout DG, Thakore PI, Moreb EA, Castellanos Rivera RM, Madhavan S, Pan X, Ran FA, Yan WX, Asokan A, Zhang F, Duan D, Gersbach CA. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science. 2016;351:403-7.
- Long C, Amoasii L, Mireault AA, McAnally JR, Li H, Sanchez-Ortiz E, Bhattacharyya S, Shelton JM, Bassel-Duby R, Olson EN. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science. 2016;351:400-3.
- Schaefer KA, Wu WH, Colgan DF, Tsang SH, Bassuk AG, Mahajan VB. Unexpected mutations after CRISPR-Cas9 editing in vivo. Nat Methods. 2017;14:547-548.