Format

Send to

Choose Destination
G3 (Bethesda). 2017 Oct 5;7(10):3533-3542. doi: 10.1534/g3.117.300123.

Single-Step qPCR and dPCR Detection of Diverse CRISPR-Cas9 Gene Editing Events In Vivo.

Author information

1
University of Pittsburgh School of Medicine, Department of Medicine, Division of Cardiology, Center for Metabolism and Mitochondrial Medicine and Vascular Medicine Institute, Pennsylvania 15261.
2
Integrated DNA Technologies, Coralville, Iowa 52241.
3
University of Pittsburgh School of Medicine, Department of Medicine, Vascular Medicine Institute, Pennsylvania 15261.
4
University of Pittsburgh School of Medicine, Department of Medicine, Division of Endocrinology, Pennsylvania 15261.
5
University of Pittsburgh School of Medicine, Department of Immunology, Pennsylvania 15261.
6
University of Pittsburgh School of Medicine, Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine, Vascular Medicine Institute, and Department of Bioengineering, Pennsylvania 15261.
7
University of Pittsburgh School of Medicine, Department of Medicine, Division of Cardiology, Center for Metabolism and Mitochondrial Medicine and Vascular Medicine Institute, Pennsylvania 15261 bkauf@pitt.edu.

Abstract

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-based technology is currently the most flexible means to create targeted mutations by recombination or indel mutations by nonhomologous end joining. During mouse transgenesis, recombinant and indel alleles are often pursued simultaneously. Multiple alleles can be formed in each animal to create significant genetic complexity that complicates the CRISPR-Cas9 approach and analysis. Currently, there are no rapid methods to measure the extent of on-site editing with broad mutation sensitivity. In this study, we demonstrate the allelic diversity arising from targeted CRISPR editing in founder mice. Using this DNA sample collection, we validated specific quantitative and digital PCR methods (qPCR and dPCR, respectively) for measuring the frequency of on-target editing in founder mice. We found that locked nucleic acid (LNA) probes combined with an internal reference probe (Drop-Off Assay) provide accurate measurements of editing rates. The Drop-Off LNA Assay also detected on-target CRISPR-Cas9 gene editing in blastocysts with a sensitivity comparable to PCR-clone sequencing. Lastly, we demonstrate that the allele-specific LNA probes used in qPCR competitor assays can accurately detect recombinant mutations in founder mice. In summary, we show that LNA-based qPCR and dPCR assays provide a rapid method for quantifying the extent of on-target genome editing in vivo, testing RNA guides, and detecting recombinant mutations.

PMID:
28860183
PMCID:
PMC5633400
DOI:
10.1534/g3.117.300123
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center