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Review
. 2015 Sep;327:102-8.
doi: 10.1016/j.heares.2015.04.016. Epub 2015 May 15.

The Application of Genome Editing in Studying Hearing Loss

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Free PMC article
Review

The Application of Genome Editing in Studying Hearing Loss

Bing Zou et al. Hear Res. .
Free PMC article

Abstract

Targeted genome editing mediated by clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) technology has emerged as one of the most powerful tools to study gene functions, and with potential to treat genetic disorders. Hearing loss is one of the most common sensory disorders, affecting approximately 1 in 500 newborns with no treatment. Mutations of inner ear genes contribute to the largest portion of genetic deafness. The simplicity and robustness of CRISPR/Cas9-directed genome editing in human cells and model organisms such as zebrafish, mice and primates make it a promising technology in hearing research. With CRISPR/Cas9 technology, functions of inner ear genes can be studied efficiently by the disruption of normal gene alleles through non-homologous-end-joining (NHEJ) mechanism. For genetic hearing loss, CRISPR/Cas9 has potential to repair gene mutations by homology-directed-repair (HDR) or to disrupt dominant mutations by NHEJ, which could restore hearing. Our recent work has shown CRISPR/Cas9-mediated genome editing can be efficiently performed in the mammalian inner ear in vivo. Thus, application of CRISPR/Cas9 in hearing research will open up new avenues for understanding the pathology of genetic hearing loss and provide new routes in the development of treatment to restore hearing. In this review, we describe major methodologies currently used for genome editing. We will highlight applications of these technologies in studies of genetic disorders and discuss issues pertaining to applications of CRISPR/Cas9 in auditory systems implicated in genetic hearing loss.

Figures

Figure 1
Figure 1
A schematic representation of programmable nucleases demonstrating two functional domains.
Figure 2
Figure 2
A schematic representation of transcriptional activator-like effector nucleases (TALENs). TALENs are composed of TALE repeats and FokI nuclease. TALENs work in paris, binding two complementary DNA strands across a spacer over which FokI nuclease dimerizes to create a double-stranded break. Each TALE repeat can recognize one single nucleotide, and multiple TALE repeats are joined in tandem to target a specific DNA sequence.
Figure 3
Figure 3
In CRISPR system, guide RNA directs Cas 9 enedonuclease to cleave the target DNA sequence 3′ bp upstream of PAM.
Figure 4
Figure 4
CRISPR/Cas9-mediated genome editing in hair cells in vivo. Cas9 protein and the GFP gRNA were complexed with the commercial lipids (Lipofectamine 2000), which were then injected into postnatal Atoh1-GFP mouse cochlea in which all outer hair cells were GFP positive. Genome editing was achieved in the outer hair cells by the absence of GFP signal in the injected inner ear, without affecting our hair cell survival. High-throughput sequencing confirmed the disruption of the GFP gene by indels. OHC: Outer Hair Cells

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