Department of Biomedical Engineering, University of Southern California, 1042 Downey Way, Los Angeles, California, 90089, USA.
Department of Bioengineering & Institute of Engineering in Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, California, 92093, USA.
Sci Rep. 2017 Jul 13;7(1):5275. doi: 10.1038/s41598-017-05722-1.
Efficient intracellular delivery of biologically active macromolecules has been a challenging but important process for manipulating live cells for research and therapeutic purposes. There have been limited transfection techniques that can deliver multiple types of active molecules simultaneously into single-cells as well as different types of molecules into physically connected individual neighboring cells separately with high precision and low cytotoxicity. Here, a high frequency ultrasound-based remote intracellular delivery technique capable of delivery of multiple DNA plasmids, messenger RNAs, and recombinant proteins is developed to allow high spatiotemporal visualization and analysis of gene and protein expressions as well as single-cell gene editing using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease (Cas9), a method called acoustic-transfection. Acoustic-transfection has advantages over typical sonoporation because acoustic-transfection utilizing ultra-high frequency ultrasound over 150 MHz can directly deliver gene and proteins into cytoplasm without microbubbles, which enables controlled and local intracellular delivery to acoustic-transfection technique. Acoustic-transfection was further demonstrated to deliver CRISPR-Cas9 systems to successfully modify and reprogram the genome of single live cells, providing the evidence of the acoustic-transfection technique for precise genome editing using CRISPR-Cas9.
高效地将生物活性大分子递送至细胞内对于研究和治疗目的的活细胞操作来说是一个具有挑战性但又十分重要的过程。目前，仅有少数转染技术能够高精度、低细胞毒性地将多种活性分子同时递送至单细胞，以及将不同类型的分子分别递送至物理连接的相邻单个细胞内。在这里，我们开发了一种基于高频超声的远程细胞内递药技术，能够递送至多种 DNA 质粒、信使 RNA 和重组蛋白，从而允许使用 CRISPR 相关蛋白-9 核酸酶 (Cas9) （一种称为声转染的方法）进行高时空可视化和基因及蛋白表达分析以及单细胞基因编辑。与典型的声孔法相比，声转染具有优势，因为利用超过 150 MHz 的超高频率超声的声转染可以直接将基因和蛋白递送至细胞质中，而无需使用微泡，这使得对声转染技术的控制和局部细胞内递药成为可能。进一步的研究表明，声转染可以成功地将 CRISPR-Cas9 系统递送至单个活细胞内以修饰和重编程基因组，为使用 CRISPR-Cas9 进行精确的基因组编辑提供了声转染技术的证据。
