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

Author:

Zhang He12,Pan Hong13,Zhou Changyang12,Wei Yu1,Ying Wenqin1,Li Shuting1ORCID,Wang Guangqin1,Li Chao1,Ren Yifei12,Li Gen456,Ding Xu456,Sun Yidi27,Li Geng-Lin4568,Song Lei456ORCID,Li Yixue27910,Yang Hui1,Liu Zhiyong1ORCID

Affiliation:

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, Guangxi, China

4. Department of Otolaryngology-Head and Neck Surgery, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

5. Ear Institute, Shanghai Jiao Tong University School of Medicine, Shanghai, China

6. Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases, Shanghai, 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, China

10. Shanghai Center for Bioinformation Technology, Shanghai Industrial Technology Institute, Shanghai, China

Abstract

In vivo genetic mutations has become a powerful tool for dissecting gene function; however, multi-gene interaction and compensatory mechanisms involving 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. The 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 firstly performed a proof-of-principle test by disrupting Atoh1, a gene critical for auditory hair cell generation. Next, we individually mutated vGlut3, Otoferlin and Prestin, three genes needed for normal hearing function. Finally, we successfully disrupted vGlut3, Otoferlin and Prestin simultaneously. Our results show that CRISPR-stop can efficiently generate single or triple homozygous F0 mice mutants, bypassing laborious mouse breeding. We believe that CRISPR-stop is a powerful method that will pave the way for high-throughput screening mouse developmental and functional genes, matching the efficiency of methods available for model organisms, such as Drosophila.

Funder

National Natural Science Foundation of China

Chinese National Science and technology

Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

Shanghai City Committee of Science and Technology

Boehringer Ingelheim International GmbH

Publisher

The Company of Biologists

Subject

Developmental Biology,Molecular Biology

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