Highlights d On-target effects (OnTEs) are present in up to 40% of human CRISPR-edited iPSC clones d OnTEs are frequently missed by standard quality controls, such as locus sequencing d Unnoticed OnTEs strongly affect phenotype formation in an iPSC Alzheimer model d Simple and broadly applicable qgPCR and SNP genotypingbased tools reliably detect OnTEs
The recent CRISPR revolution has provided researchers with powerful tools to perform genome editing in a variety of organisms. However, recent reports indicate widespread occurrence of unintended CRISPR-induced on-target effects (OnTE) at the edited site in mice and human induced pluripotent stem cells (iPSCs) that escape standard quality controls. By altering gene expression of targeted or neighboring genes, OnTE can severely affect phenotypes of CRISPR-edited cells and organisms and thus lead to data misinterpretation, which can undermine the reliability of CRISPR-based studies. Here we describe a broadly applicable framework for detecting OnTE in genome-edited cells and organisms after non-homologous end joining (NHEJ-) and homology-directed repair (HDR-)mediated editing. Our protocol enables identification of OnTE such as large deletions, large insertions, rearrangements or lossof-heterozygosity (LOH). This is achieved by subjecting genomic DNA first to quantitative genotyping PCR (qgPCR), which determines the number of intact alleles at the target site using the same PCR amplicon that has been optimized for genotyping. This combination of genotyping and quantitation allows excluding clones with mono-allelic OnTE and hemizygous editing, which are often mischaracterized as correctly edited in standard Sanger sequencing. Second, occurrence of LOH around the edited locus is detected by genotyping neighboring SNPs, using either a Sanger sequencingbased method or SNP microarrays. All steps are optimized to maximize simplicity and minimize cost to promote wide dissemination and applicability across the field. The entire protocol from genomic DNA extraction to OnTE exclusion can be performed in 6-9 days.
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 investigated the occurrence of deleterious on-target effects in human stem cells after insertion of disease-related mutations by homology-directed repair (HDR). We identified large, mono-allelic genomic deletions and loss-ofheterozygosity that escaped standard quality controls in up to 40% of edited clones. To reliably detect such events, we developed simple, low-cost and universally applicable quantitative genotyping PCR (qgPCR) as well as sequencing-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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