T158M Single Base Editing of <i>MECP2</i> Gene in Murine and Rhesus Monekey’s Embryos

doi:10.13523/j.cb.2001063

China Biotechnology

2020, Vol. 40

Issue (6): 31-39 DOI: 10.13523/j.cb.2001063

T158M Single Base Editing of MECP2 Gene in Murine and Rhesus Monekey’s Embryos

ZHOU Qin^1,²,WANG Shuang^1,²,ZHANG Ting^1,²,LI Shan-gang^1,²,CHEN Yong-chang^1,^2,^***(

)

1 College of Life Science and Technology, Institute of Primate Translational,Kunming University of Science and Technology,Kunming 650500,China
2 Yunnan Key Laboratory of Primate Biomedical Research,Kunming 650500,China

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Abstract

Objective: The T158M site of MECP2 gene was modified by using single-base editing technique to obtain single site-directed mutant rodent and non-human primate embryos,which lays a foundation for the establishment of an animal disease model simulating Rett syndrome in clinical practice.Methods: Three pairs of T158M-sgRNA plasmids were constructed using single-base editing technique,and co-transfected 293T cells with BE system(BE3 or BE4max)plasmids to screen highly-efficient sgRNAs.In vitro transcribed mRNAs were injected at different concentrations by microinjection to ICR mice and macaque fertilized eggs to measure the embryo development rate and editing efficiency of mouse and macaque and to evaluate the working efficiency of BE3 and BE4max and optimal concentration combination.Results: The mouse embryo development rate and editing efficiency are best when the concentration ratio of BE4max(ng/μl)∶sgRNA(ng/μl)on mouse embryos is 100∶50,and this concentration combination also shows effective editing efficiency on rhesus monkey embryo.Conclusion: The transition of C→T at position T158M of the MECP2 gene were successfully achieved in mouse and macaques embryos,establishing a stable embryo editing system for the later establishment of a T158M mutant RTT animal model.

Key words： Rett syndrome Single base editing Non-human primate Editing efficiency

Received: 26 January 2020 Published: 23 June 2020

ZTFLH:

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Corresponding Authors: Yong-chang CHEN E-mail: chenyc@lpbr.cn

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	Qin ZHOU
	Shuang WANG
	Ting ZHANG
	Shan-gang LI
	Yong-chang CHEN

Cite this article:

ZHOU Qin,WANG Shuang,ZHANG Ting,LI Shan-gang,CHEN Yong-chang. T158M Single Base Editing of MECP2 Gene in Murine and Rhesus Monekey’s Embryos. China Biotechnology, 2020, 40(6): 31-39.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2001063 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I6/31

Table 1 Oligo DNA designed for 158 sites

Table 2 Primers for exon 3 of MECP2/Mecp2 gene in human,rhesus monkeyand mouse

Fig.1 Construction of sgRNA plasmid (a)Oligonucleotides of sgRNA (b)Sequencing result

Fig.2 sgRNA efficiency identification on 293T cells (a) Fluorescence map of transfected cells in experimental group (b) Fluorescence of transfected cells in control group (c) Sequencing verification

Table 3 Statistics on the development of in vitro transcription of mRNAs injected into cynomolgus monkey embryos

Table 4 Developmental statistics of BE3 and BE4max in editing embryos

Table 5 BE4max system edits mouse embryo statistics on development and mutation

Fig.3 Mouse embryo validation (a) Flowchart of editing embryos and validating efficiency of BE System (b) Mouse embryo development(white arrows are embryos that have developed into blastocysts (c)T7 digestion verification (d)BE system edits the mutation type statistics of T158M locus of MECP2

Fig.4 Rhesus monkey embryo detection (a) Statistics of embryo blastocyst rates in the KO and Control groups (b) BE edited embryo wild type、 mosaic、homozygous mutation statistical results (c) Embryo development (d) Sanger sequencing analysis


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