Production of a mutant of large-scale loach Paramisgurnus dabryanus with skin pigmentation loss by genome editing with CRISPR/Cas9 system
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CRISPR/Cas9 system has been developed as a highly efficient genome editing technology to specifically induce mutations in a few aquaculture species. In this study, we described induction of targeted gene (namely tyrosinase, tyr) mutations in large-scale loach Paramisgurnus dabryanus, an important aquaculture fish species and a potential model organism for studies of intestinal air-breathing function, using the CRISPR/Cas9 system. Tyr gene in large-scale loach was firstly cloned and then its expressions were investigated. Two guide RNAs (gRNAs) were designed and separately transformed with Cas9 in the loach. 89.4% and 96.1% of injected loach juveniles respectively displayed a graded loss of pigmentation for the two gRNAs, in other words, for target 1 and target 2. We classified the injected loach juveniles into five groups according to their skin color phenotypes, including four albino groups and one wild-type-like group. And one of them was clear albino group, which was of high ornamental and commercial value. More than 50 clones for each albino transformant with a visible phenotype in each target were randomly selected and sequenced. Results obtained here showed that along with the increase of pigmentation, wild-type alleles appeared in the injected loach juveniles more often and insertion/deletion alleles less frequently. This study demonstrated that CRISPR/Cas9 system could be practically performed to modify large-scale loach tyr to produce an albino mutant of high ornamental and commercial value, and for the first time showed successful use of the CRISPR/Cas9 system for genome editing in a Cobitidae species.
KeywordsParamisgurnus dabryanus Tyrosinase gene Cloning and expression Gene knock-out CRISPR/Cas9 system Skin pigmentation loss
XX, XC and JG performed the experiments. XC designed the experiment. JG performed the bioinformatics analysis. XX and XC prepared the manuscript. All authors read and approved the final version of the manuscript.
This study was financially supported by National Key R&D Program of China (Project Number: 2018YFD0900200) and the Fundamental Research Funds for the Central Universities of China (Project Number: 2662015PY033).
Compliance with ethical standards
Conflict of interest
The authors have declared that no conflicts of interests exist.
This study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Huazhong Agricultural University. All efforts were made to minimize suffering.
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