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

• Main Authors: Laurens Raes, Melissa Pille, Aranit Harizaj, Glenn Goetgeluk, Jelter Van Hoeck, Stephan Stremersch, Juan C. Fraire, Toon Brans, Olivier Gerrit de Jong, Roel Maas-Bakker, Enrico Mastrobattista, Pieter Vader, Stefaan C. De Smedt, Bart Vandekerckhove, Koen Raemdonck, Kevin Braeckmans
• Format: Article
• Language: English
• Published: Elsevier 2021-09-01
• Series: Molecular Therapy: Nucleic Acids
• Subjects: CRISPR-Cas9; gene editing; T cells; stem cells; photoporation; intracellular delivery
• Online Access: http://www.sciencedirect.com/science/article/pii/S2162253121002080

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.