Generation and Validation of Human Pluripotent Stem Cell Disease Models Using CRISPR/Cas9 Genome Engineering

CRISPR/Cas9 mediated genome engineering of human pluripotent stem cells enables recapitulation or correction of patient-specific disease mutations allowing modelling of genetic disorders, when patient material is scarce. Disease models serve as a platform to allow detection of human specific disease mechanisms and to elucidate dysfunctional pathways. We developed a CRISPR/Cas9 genome editing protocol for hESCs and hiPSCs to efficiently generate indel based heterozygous or homozygous knockout models. This protocol includes usage of an eGFP tagged eCas9 for FACS-based single cell sorting of nucleofected stem cells to generate pure clonal lines. CRISPR mediated indels are detected by Sanger sequencing and NGS amplicon sequencing. Further genetic quality control guidelines include PCR based off-target analysis and CNV analysis. We were able to efficiently generate functional knockout lines for several genes, which play a central role for neurodevelopmental disorders such as intellectually disability-associated Coffin-Siris syndrome (SOX11), Pitt Hopkins syndrome (TCF4), and genes that were found critical in the pathophysiology of neurodegenerative disorders such as Parkinson’s disease (SNCA) and hereditary spastic paraplegia (SPG4 or SPG11). Interestingly, lines derived after treatment of some sgRNAs showed higher incident of de novo CNVs, despite no signs of off-target mutagenesis.

Currently, we are focusing on steps to optimize the efficiency for generation of corrected patient hiPSC lines by employing homology directed repair (HDR)-based mechanisms for single nucleotide substitutions. This includes testing of small molecule HDR enhancers, different sorting and analyzing strategies. Finally, we are testing novel PCR or donor based methods to generate FLAG or fluorescence marker tagged HEK293T and hPSC reporter lines.