Functional validation of mouse tyrosinase non-coding regulatory DNA elements by CRISPR-Cas9-mediated mutagenesis.
Bottom Line: Newly developed genome-editing tools, such as the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system, allow simple and rapid genetic modification in most model organisms and human cell lines.The resulting genome-edited mice showed a dramatic decrease in Tyr gene expression as inferred from the evident decrease of coat pigmentation, thus supporting the functionality of this boundary sequence in vivo, at the endogenous locus.Several potential off-targets bearing sequence similarity with each of the two RNA guides used were analyzed and found to be largely intact.
Affiliation: Department of Molecular and Cellular Biology, National Centre for Biotechnology (CNB-CSIC), Campus Cantoblanco, Darwin 3, 28049 Madrid, Spain CIBERER-ISCIII, Madrid, Spain.Show MeSH
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Mentions: The comparison of genome-edited mouse lines carrying a targeted inactivation of the 5′ Tyr boundary (this work) with other mouse transgenic lines bearing similar deletions within large chromosome-type genomic transgenes reveals interesting but fundamental differences. The absence of the Tyr 5′ boundary element in YRT4 (7), or its targeted deletion in YRT5 (7) and ΔLCR (18) YAC transgenic mice, results in a strong reduction of Tyr gene expression in neural crest-derived melanocytes, as demonstrated by a dramatic reduction in coat color pigmentation (almost albino-like), associated with strongly variegated expression (9) (Figure 5A). In contrast, the reduced coat color phenotype observed in all targeted TYRINS5 lines is milder, uniformly light grey, and without signs of variegation (Figures 4A and 5A). Additionally, a more severe phenotype is observed in the eyes of YRT4 and ΔLCR YAC transgenic mice, where a profound loss of pigment is obvious in choroidal melanocytes and strong variegation is observed at the RPE cell layer (9,18) (Figure 5C). In contrast, the expression of Tyr at the RPE cell layer appears unaffected in TYRINS5 mice in all analyzed lines, without signs of variegation, and the loss of Tyr expression in the choroid is less marked (Figure 5B), as it can also be inferred from eye melanin content measurements (Figure 4D).
Affiliation: Department of Molecular and Cellular Biology, National Centre for Biotechnology (CNB-CSIC), Campus Cantoblanco, Darwin 3, 28049 Madrid, Spain CIBERER-ISCIII, Madrid, Spain.