美国加州大学旧金山分校Alexander Marson研究团队发现聚合物稳定的Cas9纳米颗粒和修饰的修复模板提高了基因组编辑效率。2019年12月9日，《自然—生物技术》在线发表了这项成果。

研究组报告了两项改进，这些改进在临床相关的原代细胞类型中提高了基于CRISPR–Cas9的基因组编辑的效率。在同源指导的修复（HDR）模板末端添加的截短的Cas9靶序列（tCTS）与Cas9核糖核蛋白（RNP）相互作用，将模板转运至细胞核，从而将HDR效率提高了约2-4倍。此外，用多聚谷氨酸将Cas9 RNP稳定在纳米颗粒中，可进一步提高编辑效率约两倍，降低毒性，并能冻干保存而不损失活性。将这两种改进相结合，即使在减少HDR模板剂量的情况下，也可以提高基因靶向效率，在多种细胞类型（例如大体积（CD3 ⁺）T细胞、CD8 ⁺ T细胞、CD4 ⁺ T调节性T细胞（Tregs）、γδT细、B细胞、自然杀伤细胞以及原代和诱导多能干细胞衍生的造血干祖细胞（HSPC））中的多个基因组位点上产生的活编辑细胞数量大约是其两倍至六倍。

研究人员表示，需要多种多样且精确的基因组修饰以产生更广泛的过继性细胞疗法。

附：英文原文

Title: Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency

Author: 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

Issue&Volume: 2019-12-09

Abstract: Versatile and precise genome modifications are needed to create a wider range of adoptive cellular therapies1,2,3,4,5. 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).

DOI: 10.1038/s41587-019-0325-6

Source: https://www.nature.com/articles/s41587-019-0325-6