Robotic Cell Injection Surgery System for the Safe and Specific Genetic Modification in Single Cells

Genetic modification is a process by which a DNA sequence of a cell is modified, andthis process affects an organism. Genome editing is a type of genetic engineeringinvolving the insertion, deletion, or replacement of DNA in the genome of a livingorganism by using molecular scissors, such as CRISPR/Cas system. However, currentgenome editing techniques with engineered nucleases are limited in terms of safety andspecificity of these nucleases. Robotics microinjection in genetic modification will providea new way vital to address this problem, with improved throughput and success rate.In this project, a novel microsurgery robotic system will be developed, which canaccurately and quantitatively control injected materials into mammalian cells (<25µm)to genetically modify single cells with sufficient efficiency and reliability. Investigationswill be conducted on the basis of three perspectives. First, an automated microinjectionsystem that can accurately and quantitatively control injected materials into mammaliancells will be examined and created. An open architecture will be established in thesystem to perform tasks, such as cell holding, injection, and retrieval, in one completeprocess, in order to enhance processing speed and throughput. A three-dimensional (3D)image processing technique will be developed to identify and locate internal cellstructures and organelles automatically to facilitate highly efficient and accurate cellinjection surgery. Second, a microarray chip platform will be designed and fabricated toconduct single cell trapping and on-chip culture of post-injection cells. The significanceof this chip is attributed to its high trapping rate in handling small-population post-injection samples with simple and easily fabricated structures. Third, the developedmicroneedle-enabled robotic microinjection system will be applied to accurately controlthe quantity of clustered regularly interspaced short palindromic repeats/Cas9 nuclease(CRISPR/Cas9) plasmids, which are engineered nucleases for genome editing, deliveredinto each cell to generate genome-modified cells. Single cell trapping and on-chipculture of post-injection cells will be further achieved by employing the developedmicroarray chip platform to examine the influence of genetic modification on cellbiological properties.This project will provide an innovative solution to perform genome editing safely andspecifically for precision and regenerative medicine. Success of this study will facilitatethe development of new technology to enable automated cell surgery with highthroughput and success rate for advanced applications.?