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*

*Corresponding author for this work

Research output: Contribution to journalLetterpeer-review

199 Scopus citations

Abstract

Versatile and precise genome modifications are needed to create a wider range of adoptive cellular therapies1–5. 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).

Original languageEnglish
Pages (from-to)44-49
Number of pages6
JournalNature Biotechnology
Volume38
Issue number1
DOIs
StatePublished - 1 Jan 2020
Externally publishedYes

Funding

FundersFunder number
American Endowment Foundation
UCSF Biology of Infectious Diseases Training Program
National Institutes of Health
National Institute of General Medical SciencesP50GM082250
National Institute of Allergy and Infectious DiseasesT32A1007641, DP3DK111914-01, R01DK1199979
National Institute of Diabetes and Digestive and Kidney DiseasesF30DK120213
Burroughs Wellcome Fund
W. M. Keck Foundation
NATIONAL MULTIPLE SCLEROSIS SOCIETYCA 1074-A-21
Damon Runyon Cancer Research Foundation
Jeffrey Modell Foundation
Sanofi
Gilead Sciences
Center for International Studies, University of Southern CaliforniaT32 DK007418, T32GM007618
Center for Outcomes Research and Evaluation, Yale School of Medicine
Diabetes Center, University of California, San Francisco
Diabetes Research CenterNIH P30 DK063720
Division of Loan RepaymentL40 AI140341
Innovative Genomics Institute
Juno Therapeutics

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