Multiplexed Genome Editing for Efficient Phenotypic Screening in Zebrafish.

Cuizhen Zhang,Shuyu Guo,Ge Gao,Gang Peng

doi:10.3390/vetsci9020092

Cuizhen Zhang, Shuyu Guo + Show 2 more

Open Access

https://doi.org/10.3390/vetsci9020092

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Journal: Veterinary sciences	Publication Date: Feb 19, 2022
Citations: 3	License type: CC BY 4.0

Affiliation: Fudan University

Abstract

Zebrafish are widely used to investigate candidate genes for human diseases. While the emergence of CRISPR-Cas9 technology has revolutionized gene editing, the use of individual guide RNAs limits the efficiency and application of this technology in functional genetics research. Multiplexed genome editing significantly enhances the efficiency and scope of gene editing. Herein, we describe an efficient multiplexed genome editing strategy to generate zebrafish mutants. Following behavioural tests and histological examination, we identified one new candidate gene (tmem183a) for hearing loss. This study provides a robust genetic platform to quickly obtain zebrafish mutants and to identify candidate genes by phenotypic readouts.

Highlights

With the development of whole genome sequencing (WGS), whole exome sequencing (WES), and genome-wide association studies (GWAS), genetic variants in patients with human genetic diseases can be rapidly identified [1]
The candidate genes selected were: gabbr1a, gabbr2, necap1, tmem183a, and zgc103499. gabbr1a [10], gabbr2 [10,11], and necap1 [12,13] are epilepsy-associated genes, and tmem183a and zgc103499 are highly expressed in inner hair cells [14]
To maximise the indel effect, we selected gRNA target sites located at the N-terminus end of the coding sequences (Table 2), which were expected to result in premature translation terminations by indels

Summary

Introduction

With the development of whole genome sequencing (WGS), whole exome sequencing (WES), and genome-wide association studies (GWAS), genetic variants in patients with human genetic diseases can be rapidly identified [1]. Sequencing is not sufficient to distinguish true genetic variants associated with disease from broad functional variants in the human genome, and may even generate false positives [3]. This hinders the translation from genetic research to proof of clinical diagnosis, and even potentially limits the biological understanding of the disease. Autosomal recessive diseases account for a large proportion of human genetic disorders. 5% of the human genome is still without any functional annotation [4]. These genes are referred to as “dark matter genes” and there is a pressing need for functional validation. It is important to refine and expand the genetic, behavioural, and molecular toolkit in animal models

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