Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency.,Nature Biotechnology

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Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency.
Nature Biotechnology ( IF 33.1 ) Pub Date : 2019-12-09 , DOI: 10.1038/s41587-019-0325-6
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 Vo ₃ , Victoria R Tobin _{2,

3,

4} , Daniel Goodman _{2,

3,

4} , Eric Shifrut _{2,

3,

4} , Jeffrey A Bluestone _{3,

7} , Jennifer M Puck ₈ , Francis C Szoka ₉ , Alexander Marson _{1,

2,

3,

4,

10,

11,

12}

Affiliation

Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.
Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA.
Diabetes Center, University of California, San Francisco, San Francisco, CA, USA.
Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, USA.
Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
Sean N. Parker Autoimmune Research Laboratory, University of California, San Francisco, San Francisco, CA, USA.
Division of Allergy, Immunology, and Bone Marrow Transplantation, Department of Pediatrics, University of California, San Francisco, San Francisco, CA, USA.
Department of Bioengineering and Therapeutic Sciences, School of Pharmacy, University of California, San Francisco, San Francisco, CA, USA.
Chan Zuckerberg Biohub, San Francisco, CA, USA.
UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA.
Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA.

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).

中文翻译：

聚合物稳定的 Cas9 纳米颗粒和改良的修复模板提高了基因组编辑效率。

需要多功能且精确的基因组修饰来创建更广泛的过继性细胞疗法1-5。在这里，我们报告了两项改进，提高了临床相关原代细胞类型中基于 CRISPR-Cas9 的基因组编辑的效率。添加在同源定向修复 (HDR) 模板末端的截短 Cas9 靶序列 (tCTS) 与 Cas9 核糖核蛋白 (RNP) 相互作用，将模板穿梭至细胞核，从而将 HDR 效率提高约两到四倍。此外，用聚谷氨酸将 Cas9 RNP 稳定到纳米颗粒中，进一步将编辑效率提高了大约两倍，降低了毒性，并能够在不损失活性的情况下进行冻干储存。结合这两项改进，即使在减少 HDR 模板剂量的情况下，也能提高基因靶向效率，在不同细胞类型的多个基因组位点上产生大约两倍至六倍的存活编辑细胞，例如散装 (CD3+) T 细胞、CD8+ T 细胞、CD4+ T 细胞细胞、调节性 T 细胞 (Treg)、γδ T 细胞、B 细胞、自然杀伤细胞以及原代和诱导多能干细胞衍生的 6 造血干祖细胞 (HSPC)。

更新日期：2019-12-10

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