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

Overview of attention for article published in BMC Genomics, August 2016
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Title
A simplified method for identifying early CRISPR-induced indels in zebrafish embryos using High Resolution Melting analysis
Published in
BMC Genomics, August 2016
DOI 10.1186/s12864-016-2881-1
Pubmed ID
Authors

Éric Samarut, Alexandra Lissouba, Pierre Drapeau

Abstract

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. 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. 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.

Twitter Demographics

The data shown below were collected from the profile of 1 tweeter who shared this research output. Click here to find out more about how the information was compiled.

Mendeley readers

The data shown below were compiled from readership statistics for 145 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
France 1 <1%
Canada 1 <1%
Unknown 143 99%

Demographic breakdown

Readers by professional status Count As %
Student > Ph. D. Student 31 21%
Researcher 24 17%
Student > Master 22 15%
Student > Bachelor 16 11%
Student > Postgraduate 7 5%
Other 22 15%
Unknown 23 16%
Readers by discipline Count As %
Biochemistry, Genetics and Molecular Biology 48 33%
Agricultural and Biological Sciences 38 26%
Neuroscience 14 10%
Medicine and Dentistry 5 3%
Immunology and Microbiology 3 2%
Other 10 7%
Unknown 27 19%

Attention Score in Context

This research output has an Altmetric Attention Score of 1. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 07 August 2016.
All research outputs
#6,210,561
of 8,177,609 outputs
Outputs from BMC Genomics
#4,556
of 5,780 outputs
Outputs of similar age
#182,776
of 257,542 outputs
Outputs of similar age from BMC Genomics
#226
of 273 outputs
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