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Efficient introgression of allelic variants by embryo-mediated editing of the bovine genome.

Wei J, Wagner S, Lu D, Maclean P, Carlson DF, Fahrenkrug SC, Laible G - Sci Rep (2015)

Bottom Line: Next, to precisely change the LGB sequence, we co-injected ZFNs or transcription activator-like effector nucleases (TALENs) with DNA oligonucleotides (ODNs).Analysis of co-injected embryos showed targeted changes in up to 33% (ZFNs) and 46% (TALENs) of blastocysts.Deep sequence analysis of selected embryos revealed contributions of the targeted LGB allele can reach 100% which implies that genome editing by zygote injections can facilitate the one-step generation of non-mosaic livestock animals with pre-designed biallelic modifications.

View Article: PubMed Central - PubMed

Affiliation: 1] AgResearch, Ruakura, Hamilton, New Zealand [2] Guangxi University, Nabbing, China.

ABSTRACT
The recent development of designer nucleases allows for the efficient and precise introduction of genetic change into livestock genomes. Most studies so far have focused on the introduction of random mutations in cultured cells and the use of nuclear transfer to generate animals with edited genotypes. To circumvent the intrinsic uncertainties of random mutations and the inefficiencies of nuclear transfer we directed our efforts to the introduction of specific genetic changes by homology-driven repair directly in in vitro produced embryos. Initially, we injected zinc finger nuclease (ZFN)-encoding mRNA or DNA into bovine zygotes to verify cleavage activity at their target site within the gene for beta-lactoglobulin (LGB) and detected ZFN-induced random mutations in 30% to 80% of embryos. Next, to precisely change the LGB sequence, we co-injected ZFNs or transcription activator-like effector nucleases (TALENs) with DNA oligonucleotides (ODNs). Analysis of co-injected embryos showed targeted changes in up to 33% (ZFNs) and 46% (TALENs) of blastocysts. Deep sequence analysis of selected embryos revealed contributions of the targeted LGB allele can reach 100% which implies that genome editing by zygote injections can facilitate the one-step generation of non-mosaic livestock animals with pre-designed biallelic modifications.

No MeSH data available.


Deep sequencing analysis of ODN 986 co-injected embryos.Selected embryos derived from both ZFN/ODN 986 and TALENs/ODN 986 co-injections were analyzed for the presence of ODN-triggered genome edits and wild type sequencing reads. The representation of alleles with an ODN 986-specified 9 bp deletion (red) and combined wild type alleles A and B (blue) are depicted as percentage of all joined paired end sequence reads for individual embryos injected with DNA- or RNA-encoding ZFNs or TALENs as indicated. The underlying values used to generate the graph can be found in Table S2. Total percentages markedly below 100% denote the presence of additional sequence variants that are not included in the figure and are detailed in Table S1.
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f4: Deep sequencing analysis of ODN 986 co-injected embryos.Selected embryos derived from both ZFN/ODN 986 and TALENs/ODN 986 co-injections were analyzed for the presence of ODN-triggered genome edits and wild type sequencing reads. The representation of alleles with an ODN 986-specified 9 bp deletion (red) and combined wild type alleles A and B (blue) are depicted as percentage of all joined paired end sequence reads for individual embryos injected with DNA- or RNA-encoding ZFNs or TALENs as indicated. The underlying values used to generate the graph can be found in Table S2. Total percentages markedly below 100% denote the presence of additional sequence variants that are not included in the figure and are detailed in Table S1.

Mentions: Analogous to random ZFN-induced mutations, we determined the efficiency of ODN 986-mediated introgression of the specific 9 bp deletion (Fig. 4, Table S1B, Table S2). With DNA-encoded ZFNs, the frequency of the precisely edited allele was as high as 63% in embryo Z6 indicating a high homology-directed editing potential in microinjected zygotes. However, the efficiency fluctuated greatly between different embryos with some (Z4 and Z7) showing a contribution of the edited allele of below 1%. Injections of RNA-encoded ZFNs proved less successful for introgression of the deletion, as these embryos contained notably fewer mutated alleles (frequency of 0.1 to 0.6%) than their DNA-injected counterparts (Fig. 4, Table S2).


Efficient introgression of allelic variants by embryo-mediated editing of the bovine genome.

Wei J, Wagner S, Lu D, Maclean P, Carlson DF, Fahrenkrug SC, Laible G - Sci Rep (2015)

Deep sequencing analysis of ODN 986 co-injected embryos.Selected embryos derived from both ZFN/ODN 986 and TALENs/ODN 986 co-injections were analyzed for the presence of ODN-triggered genome edits and wild type sequencing reads. The representation of alleles with an ODN 986-specified 9 bp deletion (red) and combined wild type alleles A and B (blue) are depicted as percentage of all joined paired end sequence reads for individual embryos injected with DNA- or RNA-encoding ZFNs or TALENs as indicated. The underlying values used to generate the graph can be found in Table S2. Total percentages markedly below 100% denote the presence of additional sequence variants that are not included in the figure and are detailed in Table S1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496724&req=5

f4: Deep sequencing analysis of ODN 986 co-injected embryos.Selected embryos derived from both ZFN/ODN 986 and TALENs/ODN 986 co-injections were analyzed for the presence of ODN-triggered genome edits and wild type sequencing reads. The representation of alleles with an ODN 986-specified 9 bp deletion (red) and combined wild type alleles A and B (blue) are depicted as percentage of all joined paired end sequence reads for individual embryos injected with DNA- or RNA-encoding ZFNs or TALENs as indicated. The underlying values used to generate the graph can be found in Table S2. Total percentages markedly below 100% denote the presence of additional sequence variants that are not included in the figure and are detailed in Table S1.
Mentions: Analogous to random ZFN-induced mutations, we determined the efficiency of ODN 986-mediated introgression of the specific 9 bp deletion (Fig. 4, Table S1B, Table S2). With DNA-encoded ZFNs, the frequency of the precisely edited allele was as high as 63% in embryo Z6 indicating a high homology-directed editing potential in microinjected zygotes. However, the efficiency fluctuated greatly between different embryos with some (Z4 and Z7) showing a contribution of the edited allele of below 1%. Injections of RNA-encoded ZFNs proved less successful for introgression of the deletion, as these embryos contained notably fewer mutated alleles (frequency of 0.1 to 0.6%) than their DNA-injected counterparts (Fig. 4, Table S2).

Bottom Line: Next, to precisely change the LGB sequence, we co-injected ZFNs or transcription activator-like effector nucleases (TALENs) with DNA oligonucleotides (ODNs).Analysis of co-injected embryos showed targeted changes in up to 33% (ZFNs) and 46% (TALENs) of blastocysts.Deep sequence analysis of selected embryos revealed contributions of the targeted LGB allele can reach 100% which implies that genome editing by zygote injections can facilitate the one-step generation of non-mosaic livestock animals with pre-designed biallelic modifications.

View Article: PubMed Central - PubMed

Affiliation: 1] AgResearch, Ruakura, Hamilton, New Zealand [2] Guangxi University, Nabbing, China.

ABSTRACT
The recent development of designer nucleases allows for the efficient and precise introduction of genetic change into livestock genomes. Most studies so far have focused on the introduction of random mutations in cultured cells and the use of nuclear transfer to generate animals with edited genotypes. To circumvent the intrinsic uncertainties of random mutations and the inefficiencies of nuclear transfer we directed our efforts to the introduction of specific genetic changes by homology-driven repair directly in in vitro produced embryos. Initially, we injected zinc finger nuclease (ZFN)-encoding mRNA or DNA into bovine zygotes to verify cleavage activity at their target site within the gene for beta-lactoglobulin (LGB) and detected ZFN-induced random mutations in 30% to 80% of embryos. Next, to precisely change the LGB sequence, we co-injected ZFNs or transcription activator-like effector nucleases (TALENs) with DNA oligonucleotides (ODNs). Analysis of co-injected embryos showed targeted changes in up to 33% (ZFNs) and 46% (TALENs) of blastocysts. Deep sequence analysis of selected embryos revealed contributions of the targeted LGB allele can reach 100% which implies that genome editing by zygote injections can facilitate the one-step generation of non-mosaic livestock animals with pre-designed biallelic modifications.

No MeSH data available.