title: CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation.
abstract: The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) nuclease system represents an efficient tool for genome editing and gene function analysis. It consists of two components: single-guide RNA (sgRNA) and the enzyme Cas9. Typical sgRNA and Cas9 intracellular delivery techniques are limited by their reliance on cell type and exogenous materials as well as their toxic effects on cells (for example, electroporation). We introduce and optimize a microfluidic membrane deformation method to deliver sgRNA and Cas9 into different cell types and achieve successful genome editing. This approach uses rapid cell mechanical deformation to generate transient membrane holes to enable delivery of biomaterials in the medium. We achieved high delivery efficiency of different macromolecules into different cell types, including hard-to-transfect lymphoma cells and embryonic stem cells, while maintaining high cell viability. With the advantages of broad applicability across different cell types, particularly hard-to-transfect cells, and flexibility of application, this method could potentially enable new avenues of biomedical research and gene targeting therapy such as mutation correction of disease genes through combination of the CRISPR-Cas9-mediated knockin system.
authors.affiliation: Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030, USA. ; Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, NY 10065, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Han
authors.affiliation: Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030, USA. ; Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, NY 10065, USA.
authors.forename: Zongbin
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030, USA. ; Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, NY 10065, USA.
authors.forename: Myeong Chan
authors.initials: MC
authors.lastname: Jo
authors.affiliation: Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030, USA. ; Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, NY 10065, USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Zhang
authors.affiliation: Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030, USA. ; Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, NY 10065, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Pathology and Genomic Medicine and Cancer Pathology Laboratory, Houston Methodist Hospital and Houston Methodist Research Institute, Houston, TX 77030, USA.
authors.forename: Zihua
authors.initials: Z
authors.lastname: Zeng
authors.affiliation: Department of Experimental Radiation Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.
authors.forename: Nan
authors.initials: N
authors.lastname: Li
authors.affiliation: Department of Pathology and Genomic Medicine and Cancer Pathology Laboratory, Houston Methodist Hospital and Houston Methodist Research Institute, Houston, TX 77030, USA.
authors.forename: Youli
authors.initials: Y
authors.lastname: Zu
authors.affiliation: Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030, USA. ; Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, NY 10065, USA. ; Department of Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.
authors.forename: Lidong
authors.initials: L
authors.lastname: Qin
keywords: CRISPR-Cas9; Delivery; Hard-to-transfect cells; Mechanical deformation