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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.


Related in: MedlinePlus

Detection of ODN-mediated genome edits by nested PCR and restriction mapping.Shown are representative examples of PCR results for the second, mutation-specific fragment amplification from individual blastocysts that were co-injected with ZFN and ODN 970 or 986 (left hand panels) at the zygote stage. Black arrows indicate successful amplification that identifies genome-edited embryos. NC: negative control (water). The right hand panels display the result of diagnostic restriction enzyme (RE) digests of different subclones (10.2, 3, 4, 9) derived from embryo 10 co-injected with ZFN/ODN 970 and subclones 10.3 and 10.6 derived from ZFN/ODN 986 injected embryo10. The grey arrows point to diagnostic restriction fragments that differ due to the presence of a new XbaI or absence of a SfoI site in the genome edited allele. PC: positive control (wild type blastocyst) M: DNA size marker.
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f3: Detection of ODN-mediated genome edits by nested PCR and restriction mapping.Shown are representative examples of PCR results for the second, mutation-specific fragment amplification from individual blastocysts that were co-injected with ZFN and ODN 970 or 986 (left hand panels) at the zygote stage. Black arrows indicate successful amplification that identifies genome-edited embryos. NC: negative control (water). The right hand panels display the result of diagnostic restriction enzyme (RE) digests of different subclones (10.2, 3, 4, 9) derived from embryo 10 co-injected with ZFN/ODN 970 and subclones 10.3 and 10.6 derived from ZFN/ODN 986 injected embryo10. The grey arrows point to diagnostic restriction fragments that differ due to the presence of a new XbaI or absence of a SfoI site in the genome edited allele. PC: positive control (wild type blastocyst) M: DNA size marker.

Mentions: In contrast to the detection of random indels, the presence of precise ODN-mediated mutations can be readily identified by the presence of the newly introduced XbaI restriction site or by a nested PCR strategy relying on one internal primer designed to exclusively bind the mutated LGB allele. First, the exon 1 target region, potentially containing a precise ODN-induced mutation, was amplified from blastocysts derived from co-injected zygotes. The ~550 bp amplicons were then screened with a mutation-specific, nested PCR to identify genome-edited blastocysts. This revealed that co-injections of ODN 970 with DNA-encoded ZFNs efficiently generated precisely genome-edited blastocysts (Fig. 3). Template-specified mutations were detected in 18% and 33% of blastocysts derived from co-injections at 8 h and 18 h, respectively (Table 1). For these particular injections, we used variants of our standard ZFNs with plasmid constructs that expressed the ZFNs as fusion proteins with red and green fluorescent protein (ZFN-FL). Repeats of co-injections with our standard ZFNs and ODN produced comparable results (data not shown). Next, the amplified fragments from blastocysts that were positively identified for containing the precise mutation were subcloned into a plasmid vector. Individual plasmids were then subjected to Xba I restriction revealing digest patterns indicative of both mutated (two fragments of 4.2 kb and 350 bp) and wild type alleles (linearized plasmid) for different subclones of a specific blastocyst (Fig. 3). The introduced genome edits were further corroborated by sequencing subclones from a selected embryo sample. This revealed that the intended sequence changes were present with some of the subclones containing additional point mutations next to the ZFN cut site (Fig. S4A).


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)

Detection of ODN-mediated genome edits by nested PCR and restriction mapping.Shown are representative examples of PCR results for the second, mutation-specific fragment amplification from individual blastocysts that were co-injected with ZFN and ODN 970 or 986 (left hand panels) at the zygote stage. Black arrows indicate successful amplification that identifies genome-edited embryos. NC: negative control (water). The right hand panels display the result of diagnostic restriction enzyme (RE) digests of different subclones (10.2, 3, 4, 9) derived from embryo 10 co-injected with ZFN/ODN 970 and subclones 10.3 and 10.6 derived from ZFN/ODN 986 injected embryo10. The grey arrows point to diagnostic restriction fragments that differ due to the presence of a new XbaI or absence of a SfoI site in the genome edited allele. PC: positive control (wild type blastocyst) M: DNA size marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Detection of ODN-mediated genome edits by nested PCR and restriction mapping.Shown are representative examples of PCR results for the second, mutation-specific fragment amplification from individual blastocysts that were co-injected with ZFN and ODN 970 or 986 (left hand panels) at the zygote stage. Black arrows indicate successful amplification that identifies genome-edited embryos. NC: negative control (water). The right hand panels display the result of diagnostic restriction enzyme (RE) digests of different subclones (10.2, 3, 4, 9) derived from embryo 10 co-injected with ZFN/ODN 970 and subclones 10.3 and 10.6 derived from ZFN/ODN 986 injected embryo10. The grey arrows point to diagnostic restriction fragments that differ due to the presence of a new XbaI or absence of a SfoI site in the genome edited allele. PC: positive control (wild type blastocyst) M: DNA size marker.
Mentions: In contrast to the detection of random indels, the presence of precise ODN-mediated mutations can be readily identified by the presence of the newly introduced XbaI restriction site or by a nested PCR strategy relying on one internal primer designed to exclusively bind the mutated LGB allele. First, the exon 1 target region, potentially containing a precise ODN-induced mutation, was amplified from blastocysts derived from co-injected zygotes. The ~550 bp amplicons were then screened with a mutation-specific, nested PCR to identify genome-edited blastocysts. This revealed that co-injections of ODN 970 with DNA-encoded ZFNs efficiently generated precisely genome-edited blastocysts (Fig. 3). Template-specified mutations were detected in 18% and 33% of blastocysts derived from co-injections at 8 h and 18 h, respectively (Table 1). For these particular injections, we used variants of our standard ZFNs with plasmid constructs that expressed the ZFNs as fusion proteins with red and green fluorescent protein (ZFN-FL). Repeats of co-injections with our standard ZFNs and ODN produced comparable results (data not shown). Next, the amplified fragments from blastocysts that were positively identified for containing the precise mutation were subcloned into a plasmid vector. Individual plasmids were then subjected to Xba I restriction revealing digest patterns indicative of both mutated (two fragments of 4.2 kb and 350 bp) and wild type alleles (linearized plasmid) for different subclones of a specific blastocyst (Fig. 3). The introduced genome edits were further corroborated by sequencing subclones from a selected embryo sample. This revealed that the intended sequence changes were present with some of the subclones containing additional point mutations next to the ZFN cut site (Fig. S4A).

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.


Related in: MedlinePlus