Characterization of CRISPR/Cas9 RANKL knockout mesenchymal stem cell clones based on single-cell printing technology and emulsion coupling assay as a low-cellularity workflow for single-cell cloning

Abstract

The homogeneity of the genetically modified single-cells is a necessity for many applications such as cell line development, gene therapy, and tissue engineering and in particular for regenerative medical applications. The lack of tools to effectively isolate and characterize CRISPR/Cas9 engineered cells is considered as a significant bottleneck in these applications. Especially the incompatibility of protein detection technologies to confirm protein expression changes without a preconditional large-scale clonal expansion, creates a gridlock in many applications. To ameliorate the characterization of engineered cells, we propose an improved workflow, including single-cell printing/isolation technology based on fluorescent properties with high yield, a genomic edit screen (surveyor assay), mRNA rtPCR assessing altered gene expression and a versatile protein detection tool called emulsion-coupling to deliver a high-content, unified single-cell workflow. The workflow was exemplified by engineering and functionally validating RANKL knockout immortalized mesenchymal stem cells showing altered bone formation capacity of these cells. The resulting workflow is economical, without the requirement of large-scale clonal expansions of the cells with overall cloning efficiency above 30% of CRISPR/Cas9 edited cells. Nevertheless, as the single-cell clones are comprehensively characterized at an early, highly parallel phase of the development of cells including DNA, RNA, and protein levels, the workflow delivers a higher number of successfully edited cells for further characterization, lowering the chance of late failures in the development process. Author summaryI completed my undergraduate degree in biochemistry at the University of Ulm and finished my master's degree in pharmaceutical biotechnology at the University of Ulm and University of applied science of Biberach with a focus on biotechnology, toxicology and molecular biology. For my master thesis, I went to the University of Freiburg to the department of microsystems engineering, where I developed a novel workflow for cell line development. I stayed at the institute for my doctorate, but changed my scientific focus to the development of the emulsion coupling technology, which is a powerful tool for the quantitative and highly parallel measurement of protein and protein interactions. I am generally interested in being involved in the development of innovative molecular biological methods that can be used to gain new insights about biological issues. I am particularly curious to unravel the complex and often poorly understood protein interaction pathways that are the cornerstone of understanding cellular functionality and are a fundamental necessity to describe life mechanistically.