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Hybrid ssDNA repair templates enable high yield genome engineering in primary cells for disease modeling and cell therapy manufacturing

Brian R. Shy, Vivasvan Vykunta, Alvin Ha, Theodore L. Roth, Alexis Talbot, David N. Nguyen, Yan Yi Chen, Franziska Blaeschke, Shane Vedova, Murad R. Mamedov, Jing-Yi Chung, Hong Li, Jeffrey Wolf, Thomas G. Martin, Lumeng Ye, Justin Eyquem, Jonathan H. Esensten, Alexander Marson
doi: https://doi.org/10.1101/2021.09.02.458799
Brian R. Shy
1Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, USA
2Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA
3Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA
9Innovative Genomics Institute, University of California Berkeley, Berkeley, CA 94720, USA
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  • For correspondence: Alexander.Marson@ucsf.edu Brian.Shy@ucsf.edu
Vivasvan Vykunta
2Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA
3Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA
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Alvin Ha
1Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, USA
2Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA
3Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA
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Theodore L. Roth
2Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA
3Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA
5Medical Scientist Training Program, University of California San Francisco, San Francisco, CA 94143
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Alexis Talbot
1Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, USA
3Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA
7Department of Microbiology and Immunology, University of California San Francisco, San Francisco, CA 94143, USA
12Parker Institute of Cancer Immunotherapy, University of California San Francisco, San Francisco, CA, USA
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David N. Nguyen
2Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA
3Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA
9Innovative Genomics Institute, University of California Berkeley, Berkeley, CA 94720, USA
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Yan Yi Chen
2Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA
3Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA
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Franziska Blaeschke
2Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA
3Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA
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Shane Vedova
2Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA
3Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA
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Murad R. Mamedov
2Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA
3Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA
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Jing-Yi Chung
1Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, USA
3Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA
7Department of Microbiology and Immunology, University of California San Francisco, San Francisco, CA 94143, USA
12Parker Institute of Cancer Immunotherapy, University of California San Francisco, San Francisco, CA, USA
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Hong Li
11Department of Research and Development, Reagent and Services Business Unit, Life Science Group, GenScript Biotech, Nanjing, Jiangsu, China
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Jeffrey Wolf
3Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA
10UCSF Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA 94158, USA
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Thomas G. Martin
3Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA
10UCSF Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA 94158, USA
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Lumeng Ye
11Department of Research and Development, Reagent and Services Business Unit, Life Science Group, GenScript Biotech, Nanjing, Jiangsu, China
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Justin Eyquem
1Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, USA
3Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA
7Department of Microbiology and Immunology, University of California San Francisco, San Francisco, CA 94143, USA
12Parker Institute of Cancer Immunotherapy, University of California San Francisco, San Francisco, CA, USA
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Jonathan H. Esensten
1Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, USA
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Alexander Marson
2Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA
3Department of Medicine, University of California San Francisco, San Francisco, CA 94143, USA
4Institute of Human Genetics, University of California San Francisco, San Francisco, CA 94143, USA
6Chan Zuckerberg Biohub, San Francisco, CA 94158, USA
7Department of Microbiology and Immunology, University of California San Francisco, San Francisco, CA 94143, USA
8Diabetes Center, University of California San Francisco, San Francisco, CA 94143, USA
9Innovative Genomics Institute, University of California Berkeley, Berkeley, CA 94720, USA
10UCSF Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA 94158, USA
12Parker Institute of Cancer Immunotherapy, University of California San Francisco, San Francisco, CA, USA
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  • For correspondence: Alexander.Marson@ucsf.edu Brian.Shy@ucsf.edu
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Abstract

CRISPR-Cas9 offers unprecedented opportunities to modify genome sequences in primary human cells to study disease variants and reprogram cell functions for next-generation cellular therapies. CRISPR has several potential advantages over widely used retroviral vectors including: 1) site-specific transgene insertion via homology directed repair (HDR), and 2) reductions in the cost and complexity of genome modification. Despite rapid progress with ex vivo CRISPR genome engineering, many novel research and clinical applications would be enabled by methods to further improve knock-in efficiency and the absolute yield of live knock-in cells, especially with large HDR templates (HDRT). We recently reported that Cas9 target sequences (CTS) could be introduced into double-stranded DNA (dsDNA) HDRTs to improve knock-in, but yields and efficiencies were limited by toxicity at high HDRT concentrations. Here we developed a novel system that takes advantage of lower toxicity with single-stranded DNA (ssDNA). We designed hybrid ssDNA HDRTs that incorporate CTS sites and were able to boost knock-in percentages by >5-fold and live cell yields by >7-fold relative to dsDNA HDRTs with CTS. Knock-in efficiency and yield with ssCTS HDRTs were increased further with small molecule inhibitor combinations to improve HDR. We demonstrate application of these methods across a variety of target loci, knock-in constructs, and primary human cell types to reach ultra-high HDR efficiencies (>80-90%) which we use for pathogenic gene variant modeling and universal gene replacement strategies for IL2RA and CTLA4 mutations associated with mendelian immune disorders. Finally, we develop a GMP-compatible method for fully non-viral CAR-T cell manufacturing, demonstrating knock-in efficiencies of 46-62% and generating yields of >1.5 x 109 CAR+ T cells, well above current doses for adoptive cellular therapies. Taken together, we present a comprehensive non-viral approach to model disease associated mutations and re-write targeted genome sequences to program immune cell therapies at a scale compatible with future clinical application.

Competing Interest Statement

A.M. is a compensated co-founder, member of the boards of directors, and a member of the scientific advisory boards of Spotlight Therapeutics and Arsenal Biosciences. A.M. was a compensated member of the scientific advisory board at PACT Pharma and was a compensated advisor to Juno Therapeutics and Trizell. A.M. owns stock in Arsenal Biosciences, Spotlight Therapeutics, and PACT Pharma. A.M. has received fees from Merck and Vertex and is an investor in and informal advisor to Offline Ventures. The Marson lab has received research support from Juno Therapeutics, Epinomics, Sanofi, GlaxoSmithKline, Gilead, and Anthem. J.E. is a compensated co-founder at Mnemo Therapeutics. JE is a compensated scientific advisor to Cytovia Therapeutics. J.E own stocks in Mnemo Therapeutica and Cytovia Therapeutics. J.E. has received a consulting fee from Casdin Capital. The Eyquem lab has received research support from Cytovia Therapeutic and Takeda. J.E. is a holder of patents pertaining to but not resulting from this work. H.L and L.Y. are employees of Genscript Biotech Corporation. J.W. has received consulting fees from Teneobio and Adaptive Biotech. D.N.N receives consulting fees and sits on the scientific advisory board of Navan Technologies. T.L.R. is a co-founder, holds equity in, and is a member of the Scientific Advisory Board of Arsenal Bioscience. Discounted reagents were provided by Genscript. Patents have been filed based on the findings described here.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Hybrid ssDNA repair templates enable high yield genome engineering in primary cells for disease modeling and cell therapy manufacturing
Brian R. Shy, Vivasvan Vykunta, Alvin Ha, Theodore L. Roth, Alexis Talbot, David N. Nguyen, Yan Yi Chen, Franziska Blaeschke, Shane Vedova, Murad R. Mamedov, Jing-Yi Chung, Hong Li, Jeffrey Wolf, Thomas G. Martin, Lumeng Ye, Justin Eyquem, Jonathan H. Esensten, Alexander Marson
bioRxiv 2021.09.02.458799; doi: https://doi.org/10.1101/2021.09.02.458799
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Hybrid ssDNA repair templates enable high yield genome engineering in primary cells for disease modeling and cell therapy manufacturing
Brian R. Shy, Vivasvan Vykunta, Alvin Ha, Theodore L. Roth, Alexis Talbot, David N. Nguyen, Yan Yi Chen, Franziska Blaeschke, Shane Vedova, Murad R. Mamedov, Jing-Yi Chung, Hong Li, Jeffrey Wolf, Thomas G. Martin, Lumeng Ye, Justin Eyquem, Jonathan H. Esensten, Alexander Marson
bioRxiv 2021.09.02.458799; doi: https://doi.org/10.1101/2021.09.02.458799

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