Automated CRISPR/Cas9-based genome editing of human pluripotent stem cells using the StemCellFactory

Authors:
Bastian Nießing, Yannik Breitkreuz , Andreas Elanzew , Marcelo A S de Toledo , Peter Vajs , Marina Nolden , Frederik Erkens , Paul Wanek , Si Wah Christina Au Yeung , Simone Haupt , Niels König , Michael Peitz, Robert H Schmitt , Martin Zenke, Oliver Brüstle
In:
Source: Front Bioeng Biotechnol
Publication Date: (2024)
Issue: 12: 1459273
Research Area:
Stem Cells
Gene Expression
Regenerative medicine
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
Platform:
4D-Nucleofector® 96-well Systems
Experiment

3*105 hiPSCs per condition was resuspended in 20.5 µL P3 buffer and mixed with 4 µL
RNP complex. The cell suspension was transferred into a 96-well Nucleocuvette plate (Lonza) and the nucleofection was performed with a 4D-Nucleofector (Core and X Unit, Lonza) using CM150 as
program code. The optimal conditions for nucleofection, considering both efficiency and viability, were tested in advance using three different electroporation programs (CA137, DN100, and CM150). HPRT-specific gRNA (IDT), known for producing high indel rates, served as a positive control. Cell viability was assessed
using Trypan blue staining and a Neubauer counting chamber. After nucleofection, the cell suspension was directly mixed with 100 µL iPS-Brew medium supplemented with RI Y-27632 (10 µM). For culturing monoclonal single cell colonies, 2 µL cell suspension was seeded into a 6-well tissue culture plate and for the polyclonal
nucleofection controls, 40 µL cell suspension was transferred into a 24-well tissue culture plate

Abstract

CRISPR/Cas9 genome editing is a rapidly advancing technology that has the potential to accelerate research and development in a variety of fields. However, manual genome editing processes suffer from limitations in scalability, efficiency, and standardization. The implementation of automated systems for genome editing addresses these challenges, allowing researchers to cover the increasing need and perform large-scale studies for disease modeling, drug development, and personalized medicine. In this study, we developed an automated CRISPR/Cas9-based genome editing process on the StemCellFactory platform. We implemented a 4D-Nucleofector with a 96-well shuttle device into the StemCellFactory, optimized several parameters for single cell culturing and established an automated workflow for CRISPR/Cas9-based genome editing. When validated with a variety of genetic backgrounds and target genes, the automated workflow showed genome editing efficiencies similar to manual methods, with indel rates of up to 98%. Monoclonal colony growth was achieved and monitored using the StemCellFactory-integrated CellCelector, which allowed the exclusion of colonies derived from multiple cells or growing too close to neighbouring colonies. In summary, we demonstrate the successful establishment of an automated CRISPR/Cas9-based genome editing process on the StemCellFactory platform. The development of such a standardized and scalable automated CRISPR/Cas9 system represents an exciting new tool in genome editing, enhancing our ability to address a wide range of scientific questions in disease modeling, drug development and personalized medicine.