Abstract
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.
Highlights
The salamanders comprise a group of more than 700 species of amphibians and constitute the order Caudata [1]
Our results showed that the base editing technique and multiplex sgRNA strategy could efficiently induce induction of stop codons (iSTOP) to inactivate the tyr gene to create albinism phenotype
According to the principle of iSTOP, we design five sgRNAs to target the first exon of the newt tyrosinase gene (Figure 1A)
Summary
The salamanders comprise a group of more than 700 species of amphibians and constitute the order Caudata [1]. The order contains 10 families, among which are the true salamanders (family Salamandridae, including fire-belly newt, red-spotted newt, and Iberian ribbed newt) and mole salamanders (family Ambystomatidae, to which the axolotl belongs). They are historically important as biological research subjects in development, physiology, behavior, and evolution. The salamander research gained momentum in recent years with the newly assembled genomes of axolotl [3] and Iberian ribbed newt [4], as well as the ever-advancing gene and cell manipulation techniques [5]. The gold standard of gene analysis with loss- and gain-of-function procedures are inconvenient and still need to improve in salamanders
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