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G3 (Bethesda). 2015 Jan 7;5(3):407-15. doi: 10.1534/g3.114.015834.

Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases.

Author information

1
Institute of Systems and Synthetic Biology, CNRS, Université d'Evry Val d'Essonne, Evry, France.
2
Institute of Systems and Synthetic Biology, CNRS, Université d'Evry Val d'Essonne, Evry, France Nicolas.Pollet@issb.genopole.fr.

Abstract

Genome editing using engineered nucleases is used for targeted mutagenesis. But because genome editing does not target all loci with similar efficiencies, the mutation hit-rate at a given locus needs to be evaluated. The analysis of mutants obtained using engineered nucleases requires specific methods for mutation detection, and the enzyme mismatch cleavage method is used commonly for this purpose. This method uses enzymes that cleave heteroduplex DNA at mismatches and extrahelical loops formed by single or multiple nucleotides. Bacteriophage resolvases and single-stranded nucleases are used commonly in the assay but have not been compared side-by-side on mutations obtained by engineered nucleases. We present the first comparison of the sensitivity of T7E1 and Surveyor EMC assays on deletions and point mutations obtained by zinc finger nuclease targeting in frog embryos. We report the mutation detection limits and efficiencies of T7E1 and Surveyor. In addition, we find that T7E1 outperforms the Surveyor nuclease in terms of sensitivity with deletion substrates, whereas Surveyor is better for detecting single nucleotide changes. We conclude that T7E1 is the preferred enzyme to scan mutations triggered by engineered nucleases.

KEYWORDS:

CEL nucleases; T7 endonuclease I; Xenopus; enzyme mismatch cleavage; mutation screening

PMID:
25566793
PMCID:
PMC4349094
DOI:
10.1534/g3.114.015834
[Indexed for MEDLINE]
Free PMC Article

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