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A Cas9 nanoparticle system with truncated Cas9 target sequences on DNA repair templates enhances genome targeting in diverse human immune cell types
- Publication Year :
- 2019
- Publisher :
- Cold Spring Harbor Laboratory, 2019.
-
Abstract
- Virus-modified T cells are approved for cancer immunotherapy, but more versatile and precise genome modifications are needed for a wider range of adoptive cellular therapies1–4. We recently developed a non-viral CRISPR–Cas9 system for genomic site-specific integration of large DNA sequences in primary human T cells5. Here, we report two key improvements for efficiency and viability in an expanded variety of clinically-relevant primary cell types. We discovered that addition of truncated Cas9 target sequences (tCTS) at the ends of the homology directed repair (HDR) templates can interact with Cas9 ribonucleoproteins (RNPs) to ‘shuttle’ the template and enhance targeting efficiency. Further, stabilizing the Cas9 RNPs into nanoparticles with poly(glutamic acid) improved editing, reduced toxicity, and enabled lyophilized storage without loss of activity. Combining the tCTS HDR template modifications with polymer-stabilized nanoparticles increased gene targeting efficiency and viable cell yield across multiple genomic loci in diverse cell types. This system is an inexpensive, user-friendly delivery platform for non-viral genome reprogramming that we successfully applied in regulatory T cells (Tregs), γδ-T cells, B cells, NK cells, and primary and iPS-derived6 hematopoietic stem progenitor cells (HSPCs).
- Subjects :
- 0303 health sciences
Cas9
DNA repair
medicine.medical_treatment
Gene targeting
Biology
Genome
Cell biology
Homology directed repair
03 medical and health sciences
0302 clinical medicine
Cancer immunotherapy
030220 oncology & carcinogenesis
medicine
Progenitor cell
Reprogramming
030304 developmental biology
Subjects
Details
- Database :
- OpenAIRE
- Accession number :
- edsair.doi.dedup.....04e256adbe001873bc098df8a6e71f5b