Cas9 RNP transfection by vapor nanobubble photoporation for ex vivo cell engineering

Molecular Therapy: Nucleic Acids

Toon Brans¹  Jelter Van Hoeck¹  Pieter Vader¹  Bart Vandekerckhove¹  Melissa Pille¹  Roel Maas-Bakker¹  Stephan Stremersch²  Aranit Harizaj²  Kevin Braeckmans³  Koen Raemdonck³  Glenn Goetgeluk⁴  Olivier Gerrit de Jong⁴  Enrico Mastrobattista⁴  Stefaan C. De Smedt⁴  Juan C. Fraire⁴  Laurens Raes⁴ 

[1] Physical Pharmacy, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium;Department of Diagnostic Sciences, Ghent University, University Hospital Ghent, Corneel Heymanslaan 10, 9000 Ghent, Belgium;Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, the Netherlands;;Laboratory of General Biochemistry &

关键词: CRISPR-Cas9;    gene editing;    T cells;    stem cells;    photoporation;    intracellular delivery;   

DOI  :  

来源: DOAJ

【 摘 要 】

The CRISPR-Cas9 technology represents a powerful tool for genome engineering in eukaryotic cells, advancing both fundamental research and therapeutic strategies. Despite the enormous potential of the technology, efficient and direct intracellular delivery of Cas9 ribonucleoprotein (RNP) complexes in target cells poses a significant hurdle, especially in refractive primary cells. In the present work, vapor nanobubble (VNB) photoporation was explored for Cas9 RNP transfection in a variety of cell types. Proof of concept was first demonstrated in H1299-EGFP cells, before proceeding to hard-to-transfect stem cells and T cells. Gene knock-out levels over 80% and up to 60% were obtained for H1299 cells and mesenchymal stem cells, respectively. In these cell types, the unique possibility of VNB photoporation to knock out genes according to user-defined spatial patterns was demonstrated as well. Next, effective targeting of the programmed cell death 1 receptor and Wiskott-Aldrich syndrome gene in primary human T cells was demonstrated, reaching gene knock-out levels of 25% and 34%, respectively. With a throughput of >200,000 T cells per second, VNB photoporation is a scalable and versatile intracellular delivery method that holds great promise for CRISPR-Cas9-mediated ex vivo engineering of cell therapy products.

【 授权许可】

Unknown