Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency
Autor: | Linda T. Vo, P. Jonathan Li, Theodore L. Roth, Murad R. Mamedov, David N. Nguyen, Peixin Amy Chen, Eric Shifrut, Jeffrey A. Bluestone, Ryan Apathy, Alexander Marson, Francis C. Szoka, Jennifer M. Puck, Daniel B. Goodman, Victoria Tobin |
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Rok vydání: | 2019 |
Předmět: |
Adult
Polymers CD3 Biomedical Engineering Bioengineering Regenerative Medicine Applied Microbiology and Biotechnology Article 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Genome editing Stem Cell Research - Nonembryonic - Human CRISPR-Associated Protein 9 Genetics Humans Kinetoplastida Progenitor cell Induced pluripotent stem cell 030304 developmental biology Gene Editing 0303 health sciences 5.2 Cellular and gene therapies biology Protein Stability Chemistry Human Genome Polyglutamic acid Gene targeting Stem Cell Research Cell biology Haematopoiesis biology.protein RNA Nanoparticles Molecular Medicine Development of treatments and therapeutic interventions Guide 030217 neurology & neurosurgery CD8 RNA Guide Kinetoplastida Biotechnology |
Zdroj: | Nature biotechnology Nature biotechnology, vol 38, iss 1 |
ISSN: | 1546-1696 1087-0156 |
Popis: | 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). |
Databáze: | OpenAIRE |
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