Members of SCTL presented a poster at ISSCR 2020 Virtual. The poster is entitled: Chemical Cytoprotection as a Novel Strategy for Efficient and Safe Genome Editing in Human Pluripotent Stem Cells.
Authors (underlined, presenting): Hyen J. Hong, Carlos A. Tristan, Anton Simeonov, Ilyas Singeç
More information about ISSCR 2020 Virtual.
The CRISPR-Cas9 and induced pluripotent stem cell (iPSC) technologies are among the most important scientific breakthroughs and hold great promise to transform disease modeling, drug discovery, and development of new cell and gene therapies. However, low editing efficiencies and poor cell survival of dissociated iPSCs has limited the full potential of both technologies. Here, we report improved single cell cloning as well as significantly increased gene editing efficiency in human iPSCs by using a newly developed small molecule cocktail termed “CEPT” (Chen et al, 2019; bioRxiv). First, using advanced microfluidics-based cell dispensing technologies, we demonstrated that single cell cloning of iPSCs was significantly enhanced in the presence of CEPT versus the commonly used ROCK inhibitor Y-27632. Hence, identification of correctly edited clones was dramatically improved in comparison to current strategies. Second, we discovered that ordinary single cell dissociation of iPSCs caused DNA double- strand breaks as shown by the Comet assay and Western blotting against gamma-H2AX and other marker proteins. Importantly, the use of the CEPT cocktail supported the structural integrity of dissociated cells and prevented DNA damage as compared to Y-27632 or the commercially available reagent CloneR. Third, taking advantage of the cytoprotective effects conferred by CEPT, we could demonstrate increased gene editing efficiencies using various genetic and biochemical assays, thereby providing novel insights into DNA repair mechanisms in human pluripotent cells. In summary, we propose that the chemically defined CEPT cocktail will become an essential tool for improved and safer genome editing for basic research and clinical applications.