Abstract

Microfluidic Biotechnology Enables CRISPR-Cas9 System for Gene Therapy and Cancer Biomarker Discovery

Lidong Qin, Professor/CPRIT Scholar, Houston Methodist Research Institute

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) nuclease system represents an efficient tool for genome editing and gene function analysis. Integrated microfluidic chips have been well designed with micro and nanostructures to rapidly distinguish cell morphology and dynamics, which are uniquely advantageous in improving efficiency of CRISPR based delivery and screening. Here we combined the two unique technologies to facilitate cancer biomarker discovery and gene therapy. Firstly, 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. Secondly, we report a CRISPR-Cas9-mediated loss-of-function kinase screen for cancer cell deformability and invasive potential in a high-throughput microfluidic chip. In this microfluidic cell separation platform, flexible cells with high deformability and metastatic propensity were flowed out, while stiff cells remained trapped. Through deep sequencing we found that loss of certain kinases drove cells more deformable and invasive. High-ranking candidates identified included well-reported tumor suppressor kinases, such as chk2, IKK-a, p38 MAPKs and DAPK2. A high-ranking candidate STK4 was chosen for functional validation and identified to play an important role in cell deformability regulation and tumor suppression. Collectively, we have demonstrated that CRISPR-based on-chip mechanical screening is a potentially powerful strategy to facilitate systematic genetic analyses.