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Precision Engineering of the Genome, Epigenome and Transcriptome | EK24


Widespread Occurence of deleterious On-Target Effects after CRISPR Editing in human iPSCs


Mar 8, 2021 12:00am ‐ Mar 8, 2021 12:00am

Description

Widespread Occurence of deleterious On-Target Effects after CRISPR Editing in human iPSCs Isabel Weisheit1,2 , Joseph A. Kroeger1,2 , Rainer Malik1 , Julien Klimmt1,2 , Dennis Crusius1 , Angelika Dannert1, 2 , Martin Dichgans1,3 , and Dominik Paquet1,3 1 Institute for Stroke and Dementia Research (ISD), University Hospital, LMU Munich, 81377 Munich, Germany 2 Graduate School of Systemic Neurosciences, LMU Munich, 82152 Planegg-Martinsried, Germany 3 Munich Cluster for Systems Neurology (SyNergy), 81377 Munich, Germany CRISPR genome editing is a promising tool for translational research but can cause undesired editing outcomes, both on target at the edited locus and off target at other genomic loci. We investigate the occurrence of deleterious on-target effects (OnTEs) in human induced pluripotent stem cells (iPSC) after insertion of disease-related mutations by homology-directed repair (HDR) and gene editing using non-homologous end joining (NHEJ). We identify large, mono-allelic genomic deletions and loss-of-heterozygosity escaping standard quality controls in up to 40% of edited clones. Using an iPSC-based disease model, we also demonstrate deleterious and misleading consequences that unnoticed OnTE can have on experimental studies. To reliably detect such events, we describe simple, low-cost, and broadly applicable quantitative genotyping PCR (qgPCR) and single-nucleotide polymorphism (SNP) genotyping-based tools and suggest their usage as additional quality controls after editing. This will help to ensure the integrity of edited loci and increase the reliability of CRISPR editing.

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