High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails

Brian R Shy; Vivasvan S Vykunta; Alvin Ha; Alexis Talbot; Theodore L Roth; David N Nguyen; Wolfgang G Pfeifer; Yan Yi Chen; Franziska Blaeschke; Eric Shifrut; Shane Vedova; Murad R Mamedov; Jing-Yi Jing Chung; Hong Li; Ruby Yu; David Wu; Jeffrey Wolf; Thomas G Martin; Carlos E Castro; Lumeng Ye; Jonathan H Esensten; Justin Eyquem; Alexander Marson

doi:10.1038/s41587-022-01418-8

High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails

Nat Biotechnol. 2023 Apr;41(4):521-531. doi: 10.1038/s41587-022-01418-8. Epub 2022 Aug 25.

Authors

Brian R Shy^#^{1

2

3

4}, Vivasvan S Vykunta^#^{5

6}, Alvin Ha^{7

5

6}, Alexis Talbot^{5

6

8

9

10}, Theodore L Roth^{5

6

11}, David N Nguyen^{5

6

12}, Wolfgang G Pfeifer^{13

14}, Yan Yi Chen^{5

6}, Franziska Blaeschke^{5

6}, Eric Shifrut^{5

6}, Shane Vedova^{5

6}, Murad R Mamedov^{5

6}, Jing-Yi Jing Chung^{5

6

8

9}, Hong Li¹⁵, Ruby Yu^{5

6}, David Wu¹¹, Jeffrey Wolf^{6

16}, Thomas G Martin^{6

16}, Carlos E Castro^{13

17}, Lumeng Ye¹⁵, Jonathan H Esensten^{7

5}, Justin Eyquem^{5

6

8

9}, Alexander Marson^{18

19

20

21

22

23

24

25

26}

Affiliations

¹ Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, USA. Brian.Shy@ucsf.edu.
² Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA. Brian.Shy@ucsf.edu.
³ Department of Medicine, University of California San Francisco, San Francisco, CA, USA. Brian.Shy@ucsf.edu.
⁴ Innovative Genomics Institute, University of California Berkeley, Berkeley, CA, USA. Brian.Shy@ucsf.edu.
⁵ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA.
⁶ Department of Medicine, University of California San Francisco, San Francisco, CA, USA.
⁷ Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, USA.
⁸ Department of Microbiology and Immunology, University of California San Francisco, San Francisco, CA, USA.
⁹ Parker Institute for Cancer Immunotherapy, University of California San Francisco, San Francisco, CA, USA.
¹⁰ INSERM U976, Saint Louis Research Institute, Paris City University, Paris, France.
¹¹ Medical Scientist Training Program, University of California San Francisco, San Francisco, CA, USA.
¹² Innovative Genomics Institute, University of California Berkeley, Berkeley, CA, USA.
¹³ Department of Mechanical and Aerospace Engineering, The Ohio State University, Columbus, OH, USA.
¹⁴ Department of Physics, The Ohio State University, Columbus, OH, USA.
¹⁵ Department of Research and Development, Reagent and Services Business Unit, Life Science Group, GenScript Biotech, Nanjing, China.
¹⁶ UCSF Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA, USA.
¹⁷ Biophysics Graduate Program, The Ohio State University, Columbus, OH, USA.
¹⁸ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA. Alexander.Marson@ucsf.edu.
¹⁹ Department of Medicine, University of California San Francisco, San Francisco, CA, USA. Alexander.Marson@ucsf.edu.
²⁰ Innovative Genomics Institute, University of California Berkeley, Berkeley, CA, USA. Alexander.Marson@ucsf.edu.
²¹ Department of Microbiology and Immunology, University of California San Francisco, San Francisco, CA, USA. Alexander.Marson@ucsf.edu.
²² Parker Institute for Cancer Immunotherapy, University of California San Francisco, San Francisco, CA, USA. Alexander.Marson@ucsf.edu.
²³ UCSF Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA, USA. Alexander.Marson@ucsf.edu.
²⁴ Institute of Human Genetics, University of California San Francisco, San Francisco, CA, USA. Alexander.Marson@ucsf.edu.
²⁵ Chan Zuckerberg Biohub, San Francisco, CA, USA. Alexander.Marson@ucsf.edu.
²⁶ Diabetes Center, University of California San Francisco, San Francisco, CA, USA. Alexander.Marson@ucsf.edu.

^# Contributed equally.

Abstract

Enhancing CRISPR-mediated site-specific transgene insertion efficiency by homology-directed repair (HDR) using high concentrations of double-stranded DNA (dsDNA) with Cas9 target sequences (CTSs) can be toxic to primary cells. Here, we develop single-stranded DNA (ssDNA) HDR templates (HDRTs) incorporating CTSs with reduced toxicity that boost knock-in efficiency and yield by an average of around two- to threefold relative to dsDNA CTSs. Using small-molecule combinations that enhance HDR, we could further increase knock-in efficiencies by an additional roughly two- to threefold on average. Our method works across a variety of target loci, knock-in constructs and primary human cell types, reaching HDR efficiencies of >80-90%. We demonstrate application of this approach for both pathogenic gene variant modeling and gene-replacement strategies for IL2RA and CTLA4 mutations associated with Mendelian disorders. Finally, we develop a good manufacturing practice (GMP)-compatible process for nonviral chimeric antigen receptor-T cell manufacturing, with knock-in efficiencies (46-62%) and yields (>1.5 × 10⁹ modified cells) exceeding those of conventional approaches.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems* / genetics
DNA
DNA End-Joining Repair
DNA, Single-Stranded* / genetics
Gene Editing
Genome
Humans
Mutation
Recombinational DNA Repair

Substances

DNA, Single-Stranded
DNA

Abstract

Publication types

MeSH terms

Substances

Grants and funding