Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency

David N Nguyen; Theodore L Roth; P Jonathan Li; Peixin Amy Chen; Ryan Apathy; Murad R Mamedov; Linda T Vo; Victoria R Tobin; Daniel Goodman; Eric Shifrut; Jeffrey A Bluestone; Jennifer M Puck; Francis C Szoka; Alexander Marson

doi:10.1038/s41587-019-0325-6

Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency

Nat Biotechnol. 2020 Jan;38(1):44-49. doi: 10.1038/s41587-019-0325-6. Epub 2019 Dec 9.

Authors

David N Nguyen^#^{1

2

3

4}, Theodore L Roth^#^{2

3

4

5

6}, P Jonathan Li^{2

3

4}, Peixin Amy Chen^{2

3

4}, Ryan Apathy^{2

3

4}, Murad R Mamedov^{2

3

4}, Linda T Vo³, Victoria R Tobin^{2

3

4}, Daniel Goodman^{2

3

4}, Eric Shifrut^{2

3

4}, Jeffrey A Bluestone^{3

7}, Jennifer M Puck⁸, Francis C Szoka⁹, Alexander Marson^{10

11

12

13

14

15

16}

Affiliations

¹ Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.
² Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA.
³ Diabetes Center, University of California, San Francisco, San Francisco, CA, USA.
⁴ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
⁵ Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, USA.
⁶ Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
⁷ Sean N. Parker Autoimmune Research Laboratory, University of California, San Francisco, San Francisco, CA, USA.
⁸ Division of Allergy, Immunology, and Bone Marrow Transplantation, Department of Pediatrics, University of California, San Francisco, San Francisco, CA, USA.
⁹ Department of Bioengineering and Therapeutic Sciences, School of Pharmacy, University of California, San Francisco, San Francisco, CA, USA.
¹⁰ Department of Medicine, University of California, San Francisco, San Francisco, CA, USA. [email protected].
¹¹ Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA. [email protected].
¹² Diabetes Center, University of California, San Francisco, San Francisco, CA, USA. [email protected].
¹³ Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. [email protected].
¹⁴ Chan Zuckerberg Biohub, San Francisco, CA, USA. [email protected].
¹⁵ UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA. [email protected].
¹⁶ Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA. [email protected].

^# Contributed equally.

Abstract

Versatile and precise genome modifications are needed to create a wider range of adoptive cellular therapies^1-5. Here we report two improvements that increase the efficiency of CRISPR-Cas9-based genome editing in clinically relevant primary cell types. Truncated Cas9 target sequences (tCTSs) added at the ends of the homology-directed repair (HDR) template interact with Cas9 ribonucleoproteins (RNPs) to shuttle the template to the nucleus, enhancing HDR efficiency approximately two- to fourfold. Furthermore, stabilizing Cas9 RNPs into nanoparticles with polyglutamic acid further improves editing efficiency by approximately twofold, reduces toxicity, and enables lyophilized storage without loss of activity. Combining the two improvements increases gene targeting efficiency even at reduced HDR template doses, yielding approximately two to six times as many viable edited cells across multiple genomic loci in diverse cell types, such as bulk (CD3⁺) T cells, CD8⁺ T cells, CD4⁺ T cells, regulatory T cells (Tregs), γδ T cells, B cells, natural killer cells, and primary and induced pluripotent stem cell-derived⁶ hematopoietic stem progenitor cells (HSPCs).

Publication types

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

MeSH terms

Adult
CRISPR-Associated Protein 9 / metabolism*
Gene Editing
Humans
Nanoparticles / chemistry
Polymers / chemistry*
Protein Stability
RNA, Guide, CRISPR-Cas Systems / metabolism

Substances

Polymers
RNA, Guide, CRISPR-Cas Systems
CRISPR-Associated Protein 9

Abstract

Publication types

MeSH terms

Substances

Grants and funding