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Development. 2018 Oct 17;145(20). pii: dev168906. doi: 10.1242/dev.168906.

Simultaneous zygotic inactivation of multiple genes in mouse through CRISPR/Cas9-mediated base editing.

Zhang H1,2, Pan H1,3, Zhou C1,2, Wei Y1, Ying W1, Li S1, Wang G1, Li C1, Ren Y1,2, Li G4,5,6, Ding X4,5,6, Sun Y2,7, Li GL4,5,6,8, Song L4,5,6, Li Y2,7,9,10, Yang H11, Liu Z11.

Author information

1
Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
2
University of Chinese Academy of Sciences, Beijing 100049, China.
3
Guangxi University, Nanning 530004, Guangxi, China.
4
Department of Otolaryngology, Head and Neck Surgery, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.
5
Ear Institute, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China.
6
Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases, Shanghai 200011, China.
7
Key Lab of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
8
Biology Department, University of Massachusetts Amherst, Amherst, MA 01003, USA.
9
Collaborative Innovation Center for Genetics and Development, Fudan University, Shanghai 200032, China.
10
Shanghai Center for Bioinformation Technology, Shanghai Industrial Technology Institute, Shanghai 200032, China.
11
Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China huiyang@ion.ac.cn zhiyongliu@ion.ac.cn.

Abstract

In vivo genetic mutation has become a powerful tool for dissecting gene function; however, multi-gene interaction and the compensatory mechanisms involved can make findings from single mutations, at best difficult to interpret, and, at worst, misleading. Hence, it is necessary to establish an efficient way to disrupt multiple genes simultaneously. CRISPR/Cas9-mediated base editing disrupts gene function by converting a protein-coding sequence into a stop codon; this is referred to as CRISPR-stop. Its application in generating zygotic mutations has not been well explored yet. Here, we first performed a proof-of-principle test by disrupting Atoh1, a gene crucial for auditory hair cell generation. Next, we individually mutated vGlut3 (Slc17a8), otoferlin (Otof) and prestin (Slc26a5), three genes needed for normal hearing function. Finally, we successfully disrupted vGlut3, Otof and prestin simultaneously. Our results show that CRISPR-stop can efficiently generate single or triple homozygous F0 mouse mutants, bypassing laborious mouse breeding. We believe that CRISPR-stop is a powerful method that will pave the way for high-throughput screening of mouse developmental and functional genes, matching the efficiency of methods available for model organisms such as Drosophila.

KEYWORDS:

Base editing; CRISPR/Cas9; Inner ear; Otoferlin; Prestin; vGlut3

PMID:
30275281
DOI:
10.1242/dev.168906
[Indexed for MEDLINE]
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