小柯机器人

美国加州大学旧金山分校Alexander Marson和Brian R. Shy共同合作近期取得重要工作进展，他们报道使用杂合ssDNA修复模板和小分子混合物能在原代细胞中进行高效基因组编辑。相关论文2022年8月25日在线发表于《自然—生物技术》杂志上。

研究人员开发了单链DNA（ssDNA）HDR模板（HDRTs），它结合了毒性降低的CTS，相对于dsDNA CTS，敲入效率平均提高了两到三倍。使用增强HDR的小分子组合，研究人员可以进一步将敲入效率平均提高大约两到三倍。他们的方法适用于各种目标基因座、敲入构建体和人类原代细胞，HDR效率达到80–90%。研究人员展示了这种方法在与孟德尔疾病相关的IL2RA和CTLA4突变的致病基因变异建模和基因替换策略中的应用。最后，他们为非病毒嵌合抗原受体-T细胞的制造开发了一种与良好生产规范（GMP）兼容的工艺，其敲入效率（46-62%）和产量（>1.5 × 109个改良细胞）超过了传统方法。

据介绍，使用具有Cas9靶序列（CTS）的高浓度双链DNA（dsDNA）通过同源重组修复（HDR）方式，提高CRISPR介导的位点特异性基因插入效率可能对原代细胞有毒。

附：英文原文

Title: High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails

Author: Shy, Brian R., Vykunta, Vivasvan S., Ha, Alvin, Talbot, Alexis, Roth, Theodore L., Nguyen, David N., Pfeifer, Wolfgang G., Chen, Yan Yi, Blaeschke, Franziska, Shifrut, Eric, Vedova, Shane, Mamedov, Murad R., Chung, Jing-Yi Jing, Li, Hong, Yu, Ruby, Wu, David, Wolf, Jeffrey, Martin, Thomas G., Castro, Carlos E., Ye, Lumeng, Esensten, Jonathan H., Eyquem, Justin, Marson, Alexander

Issue&Volume: 2022-08-25

Abstract: Enhancing CRISPR-mediated site-specific transgene insertion efficiency by homology-directed repair (HDR) using high concentrations of double-stranded DNA (dsDNA) with Cas9 target sequences (CTSs) can be toxic to primary cells. Here, we develop single-stranded DNA (ssDNA) HDR templates (HDRTs) incorporating CTSs with reduced toxicity that boost knock-in efficiency and yield by an average of around two- to threefold relative to dsDNA CTSs. Using small-molecule combinations that enhance HDR, we could further increase knock-in efficiencies by an additional roughly two- to threefold on average. Our method works across a variety of target loci, knock-in constructs and primary human cell types, reaching HDR efficiencies of >80–90%. We demonstrate application of this approach for both pathogenic gene variant modeling and gene-replacement strategies for IL2RA and CTLA4 mutations associated with Mendelian disorders. Finally, we develop a good manufacturing practice (GMP)-compatible process for nonviral chimeric antigen receptor-T cell manufacturing, with knock-in efficiencies (46–62%) and yields (>1.5×109 modified cells) exceeding those of conventional approaches.

DOI: 10.1038/s41587-022-01418-8

Source: https://www.nature.com/articles/s41587-022-01418-8

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