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Open AccessTechnical Note

Efficient Gene Disruption via Base Editing Induced Stop in Newt Pleurodeles waltl

College of Animal Sciences and Technology, Huazhong Agricultural University, Wuhan 430070, China
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Genes 2019, 10(11), 837; https://doi.org/10.3390/genes10110837
Received: 12 September 2019 / Revised: 16 October 2019 / Accepted: 21 October 2019 / Published: 23 October 2019
(This article belongs to the Special Issue Evolutionary Genetics of Reptiles and Amphibians)
Loss-of-function approaches provide strong evidence for determining the role of particular genes. The prevalent CRISPR/Cas9 technique is widely used to disrupt target gene with uncontrolled non-homologous end joining after the double strand breaks, which results in mosaicism and multiple genotypes in the founders. In animal models with long generation time such as the salamanders, producing homozygous offspring mutants would be rather labor intensive and time consuming. Here we utilized the base editing technique to create the loss-of-function F0 mutants without the random indels. As a proof of principle, we successfully introduced premature stop codons into the tyrosinase locus and produced the albino phenotype in the newts (Pleurodeles waltl). We further demonstrated that the knockout efficiency could be greatly improved by using multiplex sgRNAs target the same gene. The F0 mutated animals showed fully loss-of-function by both genotyping and phenotyping analysis, which could enable direct functional analysis in the founders and avoid sophisticated breeding. This study not only presented the high efficiency of single base editing in a gigantic animal genome (>20 G), but also provided new tools for interrogating gene function in other salamander species. View Full-Text
Keywords: base editing; sgRNAs; newt; genome base editing; sgRNAs; newt; genome
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Cai, H.; Peng, Z.; Ren, R.; Wang, H. Efficient Gene Disruption via Base Editing Induced Stop in Newt Pleurodeles waltl. Genes 2019, 10, 837.

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