Medium-throughput in vitro detection of DNA cleavage by CRISPR-Cas12a
Quantifying DNA cleavage by CRISPR-Cas nucleases is usually done by separating the cleaved products from the non-cleaved target by agarose gel electrophoresis. We devised a method that eliminates the quantification from band intensity on agarose gel, and uses a target with a fluorescent dye on the one end and a biotin on the other. Cleavage of the target will separate the dye from the biotin, and cause the dye to stay in solution when streptavidin beads are introduced. All non-cleaved target will be eliminated from solution and no longer contribute to detectable fluorescence. Cleavage will therefore increase the fluorescent signal. A control, which has no streptavidin treatment, is taken along to correct for any errors that might have been introduced by pipetting, inactivation of the fluorescent dye or release of the biotin during several steps of the procedure. With this method we were able to quantify the fraction of active Cas12a in a purification sample and assess the cleavage rate.
| Main Authors: | , , |
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| Format: | Article/Letter to editor biblioteca |
| Language: | English |
| Subjects: | CRISPR, Cleavage, DNA, Fluorescence, Quantification, |
| Online Access: | https://research.wur.nl/en/publications/medium-throughput-in-vitro-detection-of-dna-cleavage-by-crispr-ca |
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