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BMC Genomics. 2016 Aug 4;17:547. doi: 10.1186/s12864-016-2881-1.

A simplified method for identifying early CRISPR-induced indels in zebrafish embryos using High Resolution Melting analysis.

Author information

1
Department of Neurosciences, Research Center of the University of Montreal Hospital Center (CRCHUM), Université de Montréal, Montréal, QC, Canada. eric.samarut@umontreal.ca.
2
, CRCHUM Tour Viger R09.482, 900 rue saint Denis, Montréal, QC, H2X 0A9, Canada. eric.samarut@umontreal.ca.
3
Department of Neurosciences, Research Center of the University of Montreal Hospital Center (CRCHUM), Université de Montréal, Montréal, QC, Canada.
4
Department of Pathology and Cell Biology, Université de Montréal, Montréal, QC, Canada.
5
, CRCHUM Tour Viger R09.482, 900 rue saint Denis, Montréal, QC, H2X 0A9, Canada.

Abstract

BACKGROUND:

The 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.

RESULTS:

Here 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.

CONCLUSIONS:

By 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.

KEYWORDS:

CRISPR; Genotyping; High-Resolution-Melting; Mutagenesis; Zebrafish

PMID:
27491876
PMCID:
PMC4973544
DOI:
10.1186/s12864-016-2881-1
[Indexed for MEDLINE]
Free PMC Article

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