Vouillot L et al. (2015), Comparison of T7E1 and surveyor mismatch cleava...

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G3 (Bethesda) 2015 Jan 07;53:407-15. doi: 10.1534/g3.114.015834.

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

Vouillot L , Thélie A , Pollet N .

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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.

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Species referenced: Xenopus
Genes referenced: smn1

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	Figure 1. Comparison of Surveyor and T7E1 efficiencies. Electropherograms obtained on three different DNA products (exon 2a, exon 3, and exon 6) treated using either the Surveyor (top two rows) or T7E1 enzyme (bottom two rows). The Surveyor and T7E1 assays were made on a 50% mix of wild-type and 20-bp deletion mutants. For the Surveyor and T7E1 negative controls (co-), we prepared individual reactions using only wild-type or mutant DNA and pooled the products before electrophoresis. Peaks colored in gray correspond to the internal molecular weight standards: the small molecular weight marker measures 15 bp and the large measures 1500 bp. Peaks labeled with an asterisk (*) correspond to uncleaved DNA, peaks labeled â¢ and â¬¥ correspond to cleaved DNA heteroduplex.
	Figure 2. Limits of sensitivity of Surveyor and T7E1 on different templates. Electropherograms obtained on DNA products from exon 2a, 3, and 6 after Surveyor or T7E1 digestion. The Surveyor and T7E1 assays were made on a mix composed of 5% 20-bp deletion mutants and 95% of wild-type DNA. Peaks colored in gray correspond to the internal molecular weight standards: the small molecular weight marker measures 15 bp and the large measures 1500 bp. Peaks labeled with an asterisk (*) correspond to uncleaved DNA, peaks labeled â¢ and â¬¥ correspond to cleaved DNA heteroduplex.
	Figure 3. Comparison of Surveyor and T7E1 sensitivity. Electropherograms obtained on exon 3 DNA products after Surveyor or T7E1 digestion. The percentage indicated on the right corresponds to the quantity of mutant DNA in a pool of mutant and wild-type DNA. The Surveyor and T7E1 negative controls (co-) correspond to reactions made using mutant DNA only (100%). Peaks colored in gray correspond to the internal molecular weight standards: the small molecular weight marker measures 15 bp and the large measures 1500 bp. Peaks labeled with an asterisk (*) correspond to uncleaved DNA, peaks labeled â¢ and â¬¥ correspond to cleaved DNA heteroduplex.
	Figure 4. Quantification of Surveyor and T7E1 efficiency and sensitivity. This graph shows the fraction of cleaved products from all products (y-axis) in a mixture composed of various quantities of deletion mutants of exon 3 in a population of mutant and wild-type DNA molecules (x-axis). Open circles correspond to T7E1 digestion products, open squares correspond to Surveyor digestion products.
	Figure 5. Comparison of Surveyor and T7E1 efficiencies on point mutations. The top of this figure presents schematics of the D15 and D19 sequence differences in comparison with the wild-type smn exon 2a. The electrophoregrams obtained on D15 or D19 DNA products are presented from top to bottom in the following order: after T7E1 digestion, after Surveyor digestion, and an overlay of the two conditions. The size of the peaks is given in base pairs. Only peaks called by the software were labeled. Peaks matching the size of a partial digestion product are written in bold italics. Boxed peaks are discussed in the text. The portions of the small and large markers have been omitted for clarity.

References [+] :

Babon, Improved strategy for mutation detection--a modification to the enzyme mismatch cleavage method. 1995, Pubmed