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SCIENCE CHINA Life Sciences, Volume 63 , Issue 7 : 996-1005(2020) https://doi.org/10.1007/s11427-019-1611-1

Effective generation of maternal genome point mutated porcine embryos by injection of cytosine base editor into germinal vesicle oocytes

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  • ReceivedSep 4, 2019
  • AcceptedDec 23, 2019
  • PublishedJan 21, 2020

Abstract

Cytosine and adenine base editors are promising new tools for introducing precise genetic modifications that are required to generate disease models and to improve traits in pigs. Base editors can catalyze the conversion of C→T (C>T) or A→G (A>G) in the target site through a single guide RNA. Injection of base editors into the zygote cytoplasm can result in the production of offspring with precise point mutations, but most F0 are mosaic, and breeding of F1 heterozygous pigs is time-intensive. Here, we developed a method called germinal vesicle oocyte base editing (GVBE) to produce point mutant F0 porcine embryos by editing the maternal alleles during the GV to MⅡ transition. Injection of cytosine base editor 3 (BE3) mRNA and X-linked Dmd-specific guide RNAs into GV oocytes efficiently edited maternal Dmd during in vitro maturation and did not affect the maturation potential of the oocytes. The edited MⅡ oocytes developed into blastocysts after parthenogenetic activation (PA) or in vitro fertilization (IVF). However, BE3 may reduce the developmental potential of IVF blastocysts from 31.5%±0.8% to 20.4%±2.1%. There 40%–78.3% diploid PA blastocysts had no more than two different alleles, including up to 10% embryos that had only C>T mutation alleles. Genotyping of IVF blastocysts indicated that over 70% of the edited embryos had one allele or two different alleles of Dmd. Since the male embryos had only a copy of Dmd allele, all five (5/19) F0 male embryos are homozygous and three of them were Dmd precise C>T mutation. Nine (9/19) female IVF embryos had two different alleles including a WT and a C>T mutation. DNA sequencing showed that some of them might be heterozygous embryos. In conclusion, the GVBE method is a valuable method for generating F0 embryos with maternal point mutated alleles in a single step.


Funded by

the National Key R&D Program of China(2017YFC1001901,2017YFA0102801)

the National Natural Science Foundation(31671540)

the National Transgenic Major Program(2016ZX08006003-006)

the Natural Science Foundation of Guangdong Province(2015A020212005,2014A030312011)

the Key R&D Program of Guangdong Province(2018B020203003)

and the Guangzhou Science and Technology Project(201803010020)

data collection and analysis

decision to publish

or preparation of the manuscript.


Acknowledgment

This work was supported by the National Key R&D Program of China (2017YFC1001901 and 2017YFA0102801), the National Natural Science Foundation (31671540), the National Transgenic Major Program (2016ZX08006003-006), the Natural Science Foundation of Guangdong Province (2015A020212005 and 2014A030312011), the Key R&D Program of Guangdong Province (2018B020203003), and the Guangzhou Science and Technology Project (201803010020). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


Interest statement

The author(s) declare that they have no conflict of interest.


Supplement

SUPPORTING INFORMATION

Figure S1 Sex determination of mutated embryos.

Table S1 Sequences of mutated alleles of porcine PA embryos

Table S2 Sequences of mutated alleles and sex determination of porcine IVF embryos

The supporting information is available online at http://life.scichina.com and https://link.springer.com. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


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  • Figure 1

    BE3 base editing capacity detection in porcine germinal vesicle (GV) oocytes during maturation. A, Locations and gRNA sequences of base editing targets. Four targets, according to reported human DMD mutation sites, were present in exons 20, 29, 59, and 64, respectively. B, MⅡ oocytes injected with BE3 and mScarlet mRNAs. MⅡ oocytes showing red fluorescence were picked for further analysis. C, Representative sequencing wave form of PCR product. Blue underlines indicate the gRNA sequence. Red triangles indicate base edit target sites. Green underlines indicate PAM construct. D, Comparison of different gRNA base editing efficiencies at the target site. A range of 6.8 %–54.8% MⅡ oocytes with red fluorescence were C>T mutation at the target site.

  • Figure 2

    (Color online) Developmental competence of porcine injected IVM oocytes. A and B, Cleaved and blastocyst embryos of parthenogenetic activation (PA). A, Cleaved embryos after 2 days of PA; B, Blastocysts after 7 days of PA. C and D, Cleaved and blastocyst embryos of IVF. C, Cleaved embryos after 2 days of in vitro fertilization (IVF); D, Blastocysts after 7 days of IVF. E, Statistical comparison of maturation rate and developmental rate of PA and IVF between the control and injected groups. There were no significant differences (P=0.81) in the maturation rate, cleavage rate, and blastocyst rates of PA (P=0.62 and P=0.71, respectively). The cleavage and blastocyst rates of the injected group were both significantly lower than in the control group of IVF (61.3%±3.7% vs. 85.1%±1.3%, P<0.0001; 20.4%±4.6% vs. 31.5%±8.2%, P=0.0197). Data on developmental rates are shown as the mean±SD and were analyzed by Chi-square test. PA, parthenogenetic activation; IVF, in vitro fertilization. *Because of partial oocytes losses during manipulation process, the total number of PA and IVF embryos was less than the total number of MⅡ oocytes.

  • Figure 3

    Genotyping of PA embryos of porcine oocytes injected with BE3 system at GV stage. A, Genotype schematic diagram of PA embryo. IVM, in vitro maturation; PA, parthenogenetic activation; IVC, in vitro culture. B, Dmd editing efficiency and numbers of alleles of porcine PA embryos. C, Partial sequences of mutated porcine PA embryos. *Underlined italic nucleotides in the wild-type (WT) sequence show a PAM domain. Green C of WT sequence indicates expected base editing target site. Red T and G indicate base editing products. Dark blue C>T indicates that the embryo has only the expected C>T mutation.

  • Figure 4

    Genotyping of IVF embryos of porcine oocytes injected with BE3 system at the GV stage. A, Genotype schematic diagram of IVF embryos. IVM, in vitro maturation; IVF, in vitro fertilization; IVC, in vitro culture. B, Dmd editing efficiency and numbers of alleles of porcine IVF embryos. C, Partial sequences of mutated porcine IVF embryos. *Underlined italic nucleotides in the wild-type (WT) sequence show a PAM domain. Green C of WT sequence indicates expected base editing target. Red T and G indicate base editing products. Hyphens indicate deleted nucleotides compared to those in the WT. Red small letters indicate nucleotide replacement. Dark blue C>T indicates that the embryo has only the expected C>T mutation. Brown C>T indicates that the embryo has not only the expected C>T mutation. M, male; F, female.

  • Figure 5

    Base editing frequency and accuracy of porcine oocytes injected with BE3 system at the GV stage. A, Frequency of alleles carrying on-target mutation, bystander mutation, proximal off-target mutation, and indel induced by g1, g2, and g4. Note that a single allele can carry more than one mutation. The total number of mutant alleles obtained with each base editor was set to 100%. On-target (black): target nucleotide within the 5 bp (4–8) editing window; bystander (medium grey): non-target nucleotide within the protospacer; proximal off-target (dark grey): outside the protospacer but within 200 base pairs of the target site; indel (light grey): indel induced by target site cleavage. B, Frequency of alleles carrying unanticipated products induced by g1, g2, and g4 (any mutation different from anticipated products: C>T). C, Frequency of alleles carrying only intended (anticipated) edits within the on-target region induced by g1, g2, and g4. On-target editing: any C>T mutation in the editing window; expected point editing: only predetermined (same as reported human DMD mutation sites) C>T substitution with no other mutation.

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