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Commun Biol. 2019 Feb 18;2:70. doi: 10.1038/s42003-019-0321-x. eCollection 2019.

A large CRISPR-induced bystander mutation causes immune dysregulation.

Simeonov DR1,2,3,4, Brandt AJ4,5, Chan AY3,6, Cortez JT1,2,3,4, Li Z2,3,4, Woo JM2,3,4, Lee Y2,3,4, Carvalho CMB7, Indart AC1, Roth TL1,2,3,4,5, Zou J8,9, May AP9, Lupski JR7, Anderson MS3, Buaas FW10, Rokhsar DS4,11,12, Marson A13,14,15,16,17,18.

Author information

1
Biomedical Sciences Graduate Program, University of California, San Francisco, CA, 94143, USA.
2
Department of Microbiology and Immunology, University of California, San Francisco, CA, 94143, USA.
3
Diabetes Center, University of California, San Francisco, CA, 94143, USA.
4
Innovative Genomics Institute, University of California, Berkeley, CA, 94720, USA.
5
Department of Chemistry, University of California, Berkeley, CA, 94720, USA.
6
Department of Pediatrics, University of California, San Francisco, CA, 94143, USA.
7
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, 77030, USA.
8
Department of Biomedical Data Science, Stanford University, Stanford, CA, 94305, USA.
9
Chan Zuckerberg Biohub, San Francisco, CA, 94158, USA.
10
Genetic Engineering Technologies, The Jackson Laboratory, Bar Harbor, ME, 04609, USA.
11
Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720, USA.
12
Okinawa Institute of Science and Technology, Okinawa, 904-0495, Japan.
13
Department of Microbiology and Immunology, University of California, San Francisco, CA, 94143, USA. alexander.marson@ucsf.edu.
14
Diabetes Center, University of California, San Francisco, CA, 94143, USA. alexander.marson@ucsf.edu.
15
Innovative Genomics Institute, University of California, Berkeley, CA, 94720, USA. alexander.marson@ucsf.edu.
16
Chan Zuckerberg Biohub, San Francisco, CA, 94158, USA. alexander.marson@ucsf.edu.
17
Department of Medicine, University of California, San Francisco, CA, 94143, USA. alexander.marson@ucsf.edu.
18
UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, 94158, USA. alexander.marson@ucsf.edu.

Abstract

A persistent concern with CRISPR-Cas9 gene editing has been the potential to generate mutations at off-target genomic sites. While CRISPR-engineering mice to delete a ~360 bp intronic enhancer, here we discovered a founder line that had marked immune dysregulation caused by a 24 kb tandem duplication of the sequence adjacent to the on-target deletion. Our results suggest unintended repair of on-target genomic cuts can cause pathogenic "bystander" mutations that escape detection by routine targeted genotyping assays.

Conflict of interest statement

A.M. is a co-founder of Spotlight Therapeutics, Arsenal Biosciences, and Sonoma Biotherapeutics. D.R.S. is a co-founder of Beeline Therapeutics. A.M. has served as an advisor to Juno Therapeutics and is a member of the scientific advisory board for PACT Pharma. The Marson lab has received sponsored research support from Juno Therapeutics, Epinomics and Sanofi and a gift from Gilead. The Marson lab has received funding from the Parker Institute for Cancer Immunotherapy (PICI) and the Innovative Genomics Institute (IGI). Baylor College of Medicine (BCM) and Miraca Holdings have formed a joint venture with shared ownership and governance of the Baylor Genetics (BG), which performs clinical microarray analysis and clinical exome sequencing. J.R.L. serves on the Scientific Advisory Board of the BG. J.R.L. has stock ownership in 23andMe, is a paid consultant for Regeneron Pharmaceuticals, and is a co-inventor on multiple United States and European patents related to molecular diagnostics for inherited neuropathies, eye diseases, and bacterial genomic fingerprinting. The remaining authors declare no competing interests.

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