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Simple and reliable detection of CRISPR-induced on-target effects by qgPCR and SNP genotyping


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 (OnTEs) 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 neighbouring genes, OnTEs 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 OnTEs in genome-edited cells and organisms after non-homologous end joining-mediated and homology-directed repair-mediated editing. Our protocol enables identification of OnTEs such as large deletions, large insertions, rearrangements or loss of 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 makes it possible to exclude clones with monoallelic OnTEs 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 neighbouring single-nucleotide polymorphisms (SNPs), using either a Sanger sequencing-based 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 d.

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Fig. 1: CRISPR–Cas9 genome editing induces deleterious OnTEs that can be detected by qgPCR and SNP genotyping in a simple and reliable manner.
Fig. 2: Overview of the procedure for simple and reliable OnTE detection after CRISPR genome editing.
Fig. 3: Anticipated results for OnTE detection—example of CRISPR editing at the APPSwe locus in human iPSCs.

Data availability

Data that support the findings of this study are available in Mendeley Data,


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This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy within the framework of the Munich Cluster for Systems Neurology (EXC 2145 SyNergy—ID 390857198), Vascular Dementia Research Foundation, VERUM Foundation, Wilhelm-Vaillant-Foundation, and the donors of the ADR AD2019604S, a program of the BrightFocus Foundation (to D.P.), and Helmholtz Association ‘ExNet-0041-Phase2-3 (‘SyNergy-HMGU’)’, Else Kröner Fresenius Stiftung (ForTra-gGmbH; genome editing, to W.W.). We also thank ISAR Bioscience GmbH for technical help with performance of RNP editing experiments.

Author information

Authors and Affiliations



Conceptualization, I.W. and D.P.; methodology, I.W., J.A.K, R.M., B.W., P.L. and D.P.; writing—original draft, I.W., J.A.K., R.M., B.W., P.L. and D.P.; writing—review and editing, I.W., J.A.K., R.M., B.W., P.L., W.W., M.D. and D.P.; funding acquisition, W.W., M.D. and D.P.; supervision, D.P.

Corresponding author

Correspondence to Dominik Paquet.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Marco Herold, Chengyu Liu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key reference using this protocol

Weisheit, I. et al. Cell Rep. 31, 107689 (2020):

Supplementary information

Supplementary Table 1

Primer, sgRNA and ssODN sequences

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Weisheit, I., Kroeger, J.A., Malik, R. et al. Simple and reliable detection of CRISPR-induced on-target effects by qgPCR and SNP genotyping. Nat Protoc 16, 1714–1739 (2021).

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