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An efficient platform for generating somatic point mutations with germline transmission in the zebrafish by CRISPR/Cas9-mediated gene editing
Yibo Zhang1; Zhiwei Zhang1; Wei Ge2
2018-03-02
Source PublicationJOURNAL OF BIOLOGICAL CHEMISTRY
ISSN0021-9258
Volume293Issue:17Pages:6611-6622
Abstract

Homology-directed recombination (HDR)-mediated genome editing is a powerful approach for both basic functional study and disease modeling. Although some studies have reported HDR-mediated precise editing in nonrodent models, the efficiency of establishing pure mutant animal lines that carry specific amino acid substitutions remains low. Furthermore, because the efficiency of nonhomologous end joining (NHEJ)-induced insertion and deletion (indel) mutations is normally much higher than that of HDR-induced point mutations, it is often difficult to identify the latter in the background of indel mutations. Using zebrafish as the model organism and Y box-binding protein 1 (Ybx1/ybx1) as the model molecule, we have established an efficient platform for precise CRISPR/Cas9-mediated gene editing in somatic cells, yielding an efficiency of up to 74% embryos. Moreover, we established a procedure for screening germline transmission of point mutations out of indel mutations even when germline transmission efficiency was low (<2%). To further improve germline transmission of HDR-induced point mutations, we optimized several key factors that may affect HDR efficiency, including the type of DNA donor, suppression of NHEJ, stimulation of HDR pathways, and use of Cas9 protein instead of mRNA. The optimized combination of these factors significantly increased the efficiency of germline transmission of point mutation up to 25%. In summary, we have developed an efficient procedure for creating point mutations and differentiating mutant individuals from those carrying knockouts of entire genes.

KeywordSomatic Point Mutation Crispr/cas9 Homology-directed Recombination (Hdr) Knock-in Zebrafish
DOIhttp://doi.org/10.1074/jbc.RA117.001080
URLView the original
Indexed BySCI
Language英语
WOS Research AreaBiochemistry & Molecular Biology
WOS SubjectBiochemistry & Molecular Biology
WOS IDWOS:000431108600031
PublisherAMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
The Source to ArticleWOS
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Cited Times [WOS]:10   [WOS Record]     [Related Records in WOS]
Document TypeJournal article
CollectionFaculty of Health Sciences
Corresponding AuthorWei Ge
Affiliation1.From the Centre of Reproduction, Development and Aging (CRDA), Faculty of Health Sciences, University of Macau, Macau 999078, China.
2.From the Centre of Reproduction, Development and Aging (CRDA), Faculty of Health Sciences, University of Macau, Macau 999078, China
First Author AffilicationFaculty of Health Sciences
Corresponding Author AffilicationFaculty of Health Sciences
Recommended Citation
GB/T 7714
Yibo Zhang,Zhiwei Zhang,Wei Ge. An efficient platform for generating somatic point mutations with germline transmission in the zebrafish by CRISPR/Cas9-mediated gene editing[J]. JOURNAL OF BIOLOGICAL CHEMISTRY,2018,293(17):6611-6622.
APA Yibo Zhang,Zhiwei Zhang,&Wei Ge.(2018).An efficient platform for generating somatic point mutations with germline transmission in the zebrafish by CRISPR/Cas9-mediated gene editing.JOURNAL OF BIOLOGICAL CHEMISTRY,293(17),6611-6622.
MLA Yibo Zhang,et al."An efficient platform for generating somatic point mutations with germline transmission in the zebrafish by CRISPR/Cas9-mediated gene editing".JOURNAL OF BIOLOGICAL CHEMISTRY 293.17(2018):6611-6622.
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