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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.


LGB target locus and target-specific indels generated by ZFN injection into one cell embryos.(A) Shown is the DNA sequence for the relevant region of the LGB target locus for the two main wild type LGB variants A (WT-A) and B (WT-B). The encoded amino acids of BLG are given in single letter code above the DNA sequence with lower case indicating amino acids of the signal peptide and upper case amino acids of the mature protein. Nucleotides highlighted in green depict polymorphic sites differing in WT-A and WT-B. The ZFN binding sites are indicated by grey boxes and the ZFN cleavage site is underlined. (B) Sequence of the TaqMan probe used for the mutation specific PCR assay and its position relative to the LGB locus. At the polymorphic site, the probe contains a pyrimidine (either C or T) shown as Y. (C) Sequence of mutated alleles generated by injection of DNA-encoded ZFNs into individual IVF embryos, numbered on the left. The mutations are detailed on the right with size in bp, deletion (DEL), insertion (INS), transversions (TV) and transitions (TS) and in which LGB variant (A or B) the mutation was introduced. Sequence changes of DELs are indicated as dash and additional nucleotides and other point mutations are highlighted in red. D) Mutations generated by injection of RNA-encoded ZFNs into individual IVF embryos.
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f2: LGB target locus and target-specific indels generated by ZFN injection into one cell embryos.(A) Shown is the DNA sequence for the relevant region of the LGB target locus for the two main wild type LGB variants A (WT-A) and B (WT-B). The encoded amino acids of BLG are given in single letter code above the DNA sequence with lower case indicating amino acids of the signal peptide and upper case amino acids of the mature protein. Nucleotides highlighted in green depict polymorphic sites differing in WT-A and WT-B. The ZFN binding sites are indicated by grey boxes and the ZFN cleavage site is underlined. (B) Sequence of the TaqMan probe used for the mutation specific PCR assay and its position relative to the LGB locus. At the polymorphic site, the probe contains a pyrimidine (either C or T) shown as Y. (C) Sequence of mutated alleles generated by injection of DNA-encoded ZFNs into individual IVF embryos, numbered on the left. The mutations are detailed on the right with size in bp, deletion (DEL), insertion (INS), transversions (TV) and transitions (TS) and in which LGB variant (A or B) the mutation was introduced. Sequence changes of DELs are indicated as dash and additional nucleotides and other point mutations are highlighted in red. D) Mutations generated by injection of RNA-encoded ZFNs into individual IVF embryos.

Mentions: For our study we first used a ZFN pair which binds to a target sequence in exon 1 of the bovine LGB locus and cleaves within the sequence encoding amino acids four and five of the mature BLG protein (Fig. 2A). The ZFN pair was designed to target one of the two main wild type alleles, variant B which differs from variant A by three SNPs within the target region, one of which is located within the binding site for the 5’ ZFN monomer (Fig. 2A). The ZFNs had been previously activity-verified for the successful introduction of indels in both LGB variants in primary bovine cells10. To determine whether these ZFNs could also induce mutations in bovine embryos produced by in vitro fertilization (IVF), we co-injected plasmid DNA or in vitro-transcribed polyadenylated RNA encoding the ZFN pair and GFP RNA into the cytoplasm of zygotes. We chose to inject at two different time points: 8 h (immediately after completion of IVF) and 18 h (successfully used previously11) post fertilization, reasoning that an earlier time point for injections may result in earlier expression and avoid or limit the generation of mosaic embryos. About 11% to 12% of zygotes co-injected with ZFN DNA and GFP RNA developed to blastocysts (Table 1). Co-injections using RNA-encoded ZFNs in combination with GFP RNA showed slightly better development rates with about one third of injected zygotes developing into blastocysts. GFP expression proved to be an unreliable reporter for indirectly indicating ZFN expression in co-injected embryos. While uniform GFP expression was readily detectable in most or all blastocysts in some experiments (Fig. S1), in other co-injection experiments none of the blastocysts showed detectable GFP expression even though most of these blastocysts carried ZFN-triggered mutations (Table 1). Blastocysts developed from injected zygotes were analyzed for random, ZFN-triggered mutations at the ZFN cut site, initially with the most commonly used Cel-I (Transgenomic)12 or T7E1 (New England Biolabs)13 mismatch cleavage assays. In both assays, detection of cleavage products indicates the presence of ZFN-induced mutations at the target site. We did, however, consistently observe high background levels of mismatch cleavage bands in samples of control blastocysts developed from non-injected zygotes (data not shown), which may be due to mismatch cleavage in “LGB heterozygous” embryos containing wild type allele variants A and B. Because this rendered the results unreliable, we alternatively developed a TaqMan PCR assay with a TaqMan probe designed to only bind the known wild type sequence (Fig. 2B) while the occurrence of indels or SNPs in or near the ZFN cut site prevents binding of the probe. With the expectation that blastocysts derived from injected zygotes will carry variable ratios of wild type and mutated alleles, fragments of the LGB target locus were amplified from blastocysts and subcloned into a plasmid prior to analysis. Following transformation into bacteria, individual bacterial colonies were analyzed with the TaqMan assay and melt temperature assay of the PCR amplicons to identify the presence of genome-edited sequences (Fig. S2). Analysis of a total of thirty IVF blastocysts, co-injected at the zygote stage, revealed that a high percentage of blastocysts (including GFP-negative blastocysts), ranging from 29% to 83% for RNA and DNA injections at the two time points, carried mutations (Table 1). None of the treatment groups appeared to offer superior efficiencies with no significant differences being detectable between the different experimental conditions.


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)

LGB target locus and target-specific indels generated by ZFN injection into one cell embryos.(A) Shown is the DNA sequence for the relevant region of the LGB target locus for the two main wild type LGB variants A (WT-A) and B (WT-B). The encoded amino acids of BLG are given in single letter code above the DNA sequence with lower case indicating amino acids of the signal peptide and upper case amino acids of the mature protein. Nucleotides highlighted in green depict polymorphic sites differing in WT-A and WT-B. The ZFN binding sites are indicated by grey boxes and the ZFN cleavage site is underlined. (B) Sequence of the TaqMan probe used for the mutation specific PCR assay and its position relative to the LGB locus. At the polymorphic site, the probe contains a pyrimidine (either C or T) shown as Y. (C) Sequence of mutated alleles generated by injection of DNA-encoded ZFNs into individual IVF embryos, numbered on the left. The mutations are detailed on the right with size in bp, deletion (DEL), insertion (INS), transversions (TV) and transitions (TS) and in which LGB variant (A or B) the mutation was introduced. Sequence changes of DELs are indicated as dash and additional nucleotides and other point mutations are highlighted in red. D) Mutations generated by injection of RNA-encoded ZFNs into individual IVF embryos.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: LGB target locus and target-specific indels generated by ZFN injection into one cell embryos.(A) Shown is the DNA sequence for the relevant region of the LGB target locus for the two main wild type LGB variants A (WT-A) and B (WT-B). The encoded amino acids of BLG are given in single letter code above the DNA sequence with lower case indicating amino acids of the signal peptide and upper case amino acids of the mature protein. Nucleotides highlighted in green depict polymorphic sites differing in WT-A and WT-B. The ZFN binding sites are indicated by grey boxes and the ZFN cleavage site is underlined. (B) Sequence of the TaqMan probe used for the mutation specific PCR assay and its position relative to the LGB locus. At the polymorphic site, the probe contains a pyrimidine (either C or T) shown as Y. (C) Sequence of mutated alleles generated by injection of DNA-encoded ZFNs into individual IVF embryos, numbered on the left. The mutations are detailed on the right with size in bp, deletion (DEL), insertion (INS), transversions (TV) and transitions (TS) and in which LGB variant (A or B) the mutation was introduced. Sequence changes of DELs are indicated as dash and additional nucleotides and other point mutations are highlighted in red. D) Mutations generated by injection of RNA-encoded ZFNs into individual IVF embryos.
Mentions: For our study we first used a ZFN pair which binds to a target sequence in exon 1 of the bovine LGB locus and cleaves within the sequence encoding amino acids four and five of the mature BLG protein (Fig. 2A). The ZFN pair was designed to target one of the two main wild type alleles, variant B which differs from variant A by three SNPs within the target region, one of which is located within the binding site for the 5’ ZFN monomer (Fig. 2A). The ZFNs had been previously activity-verified for the successful introduction of indels in both LGB variants in primary bovine cells10. To determine whether these ZFNs could also induce mutations in bovine embryos produced by in vitro fertilization (IVF), we co-injected plasmid DNA or in vitro-transcribed polyadenylated RNA encoding the ZFN pair and GFP RNA into the cytoplasm of zygotes. We chose to inject at two different time points: 8 h (immediately after completion of IVF) and 18 h (successfully used previously11) post fertilization, reasoning that an earlier time point for injections may result in earlier expression and avoid or limit the generation of mosaic embryos. About 11% to 12% of zygotes co-injected with ZFN DNA and GFP RNA developed to blastocysts (Table 1). Co-injections using RNA-encoded ZFNs in combination with GFP RNA showed slightly better development rates with about one third of injected zygotes developing into blastocysts. GFP expression proved to be an unreliable reporter for indirectly indicating ZFN expression in co-injected embryos. While uniform GFP expression was readily detectable in most or all blastocysts in some experiments (Fig. S1), in other co-injection experiments none of the blastocysts showed detectable GFP expression even though most of these blastocysts carried ZFN-triggered mutations (Table 1). Blastocysts developed from injected zygotes were analyzed for random, ZFN-triggered mutations at the ZFN cut site, initially with the most commonly used Cel-I (Transgenomic)12 or T7E1 (New England Biolabs)13 mismatch cleavage assays. In both assays, detection of cleavage products indicates the presence of ZFN-induced mutations at the target site. We did, however, consistently observe high background levels of mismatch cleavage bands in samples of control blastocysts developed from non-injected zygotes (data not shown), which may be due to mismatch cleavage in “LGB heterozygous” embryos containing wild type allele variants A and B. Because this rendered the results unreliable, we alternatively developed a TaqMan PCR assay with a TaqMan probe designed to only bind the known wild type sequence (Fig. 2B) while the occurrence of indels or SNPs in or near the ZFN cut site prevents binding of the probe. With the expectation that blastocysts derived from injected zygotes will carry variable ratios of wild type and mutated alleles, fragments of the LGB target locus were amplified from blastocysts and subcloned into a plasmid prior to analysis. Following transformation into bacteria, individual bacterial colonies were analyzed with the TaqMan assay and melt temperature assay of the PCR amplicons to identify the presence of genome-edited sequences (Fig. S2). Analysis of a total of thirty IVF blastocysts, co-injected at the zygote stage, revealed that a high percentage of blastocysts (including GFP-negative blastocysts), ranging from 29% to 83% for RNA and DNA injections at the two time points, carried mutations (Table 1). None of the treatment groups appeared to offer superior efficiencies with no significant differences being detectable between the different experimental conditions.

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.