Abstract
BackgroundThe CRISPR/Cas9 system has become a regularly used tool for editing the genome of many model organisms at specific sites. However, two limiting steps arise in the process of validating guide RNA target sites in larvae and adults: the time required to identify indels and the cost associated with identifying potential mutant animals.ResultsHere we have combined and optimized the HotSHOT genomic DNA extraction technique with a two-steps Evagreen PCR, followed by a high-resolution melting (HRM) assay, which facilitates rapid identification of CRISPR-induced indels. With this technique, we were able to genotype adult zebrafish using genomic DNA extracted from fin-clips in less than 2 h. We were also able to obtain a reliable and early read-out of the effectiveness of guide RNAs only 4 h after the embryos were injected with the constructs for the CRISPR/Cas9 mutagenic system. Furthermore, through mutagenesis kinetic assay, we identified that the 2-cell stage is the earliest time point at which indels can be observed.ConclusionsBy combining an inexpensive and rapid genomic DNA extraction method with an HRM-based assay, our approach allows for high-throughput genotyping of adult zebrafish and embryos, and is more sensitive than standard PCR approaches, permitting early identification of CRISPR-induced indels and with applications for other model organisms as well.
Highlights
The Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated endonuclease 9 (Cas9) system has become a regularly used tool for editing the genome of many model organisms at specific sites
Standard methods generally assess the efficacy of CRISPR/Cas9 mutagenesis by extracting DNA from a 24-h embryo and indels are usually detected by T7E1 endonuclease assay, restriction enzyme screening or fluorescent PCR [6,7,8,9]
HotSHOT genomic DNA extraction method is suitable for high-resolution melting (HRM) analysis Our aim was to establish a reliable and cost-effective method for genotyping zebrafish embryos subjected to CRISPR/cas9 mutagenesis that could be used as a precise analysis tool for a broad range of applications
Summary
The CRISPR/Cas system has become a regularly used tool for editing the genome of many model organisms at specific sites. Commonly used genotyping techniques are based on a locus-specific amplification by PCR followed by amplicon digestion of a specific restriction enzyme or sequencing. This requires adequate genomic DNA for the reaction and substantial time to complete the screen. We report the combination of the HotSHOT raw genomic extraction method followed by a HRM analysis for genotyping both adult zebrafish and individual embryos. This optimized protocol allows fast (within two hours) and Samarut et al BMC Genomics (2016) 17:547 cost-efficient adult genotyping. Our results show that CRISPR-induced mutations are detectable as early as after the first cell division of the embryo
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