Functional validation of mouse tyrosinase non-coding regulatory DNA elements by CRISPR–Cas9-mediated mutagenesis

Abstract

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. Here, we report the production and analysis of mice carrying the inactivation via deletion of a genomic insulator, a key non-coding regulatory DNA element found 5′ upstream of the mouse tyrosinase (Tyr) gene. Targeting sequences flanking this boundary in mouse fertilized eggs resulted in the efficient deletion or inversion of large intervening DNA fragments delineated by the RNA guides. 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. This study reports how non-coding DNA elements, even if located in repeat-rich genomic sequences, can be efficiently and functionally evaluated in vivo and, furthermore, it illustrates how the regulatory elements described by the ENCODE and EPIGENOME projects, in the mouse and human genomes, can be systematically validated.

A CRISPR Path to Engineering New Genetic Mouse Models for Cardiovascular Research

“…In this context, an insulator located within repeat sequences, and hence intractable for removal through conventional gene targeting, was deleted with 2c CRISPR; mice lacking the regulatory region exhibited reduced expression of the Tyr gene. 102 …”

Functional interrogation of non-coding DNA through CRISPR genome editing

“…Notably, targeted deletion has been used to delete composite enhancers as well as enhancer substituents to identify functional sequence within large enhancer regions with characteristic epigenetic marks [63–65]. This strategy has also been used to generate genomic inversions with inverted sequences of greater than 1 megabase [33,55,66–69]. For example, inversion of CTCF binding sites induced dysregulation of genomic topology and alteration of enhancer-promoter contacts, which suggest that enhancers containing CTCF binding sites may not display the orientation independence traditionally ascribed to enhancer elements [70].…”

Genome Editing of Structural Variations: Modeling and Gene Correction

“…According to a recent report, F0 chimera mice with SVs, including inversions, can be prepared within approximately 10 weeks using the CRISPR/ Cas9 system, which is much faster than conventional methods [41]. Thus, the combination of the in vivo mouse system and the Cas9 system can help us to understand SVs more quickly and may be an important tool for finding key DNA elements in protein non-coding regions, and for studying the effect of topological chromatin domains on the expression and function of specific genes [42,43]. Inversions using the RGEN system were also successfully induced by microinjection into the silkworm and zebrafish embryos [44,45].…”