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Efficient nonmeiotic allele introgression in livestock using custom endonucleases.

ABSTRACT: We have expanded the livestock gene editing toolbox to include transcription activator-like (TAL) effector nuclease (TALEN)- and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-stimulated homology-directed repair (HDR) using plasmid, rAAV, and oligonucleotide templates. Toward the genetic dehorning of dairy cattle, we introgressed a bovine POLLED allele into horned bull fibroblasts. Single nucleotide alterations or small indels were introduced into 14 additional genes in pig, goat, and cattle fibroblasts using TALEN mRNA and oligonucleotide transfection with efficiencies of 10-50% in populations. Several of the chosen edits mimic naturally occurring performance-enhancing or disease- resistance alleles, including alteration of single base pairs. Up to 70% of the fibroblast colonies propagated without selection harbored the intended edits, of which more than one-half were homozygous. Edited fibroblasts were used to generate pigs with knockout alleles in the DAZL and APC genes to model infertility and colon cancer. Our methods enable unprecedented meiosis-free intraspecific and interspecific introgression of select alleles in livestock for agricultural and biomedical applications.

SUBMITTER: Tan W

PROVIDER: S-EPMC3799378 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Efficient nonmeiotic allele introgression in livestock using custom endonucleases.

Tan Wenfang W Carlson Daniel F DF Lancto Cheryl A CA Garbe John R JR Webster Dennis A DA Hackett Perry B PB Fahrenkrug Scott C SC

Proceedings of the National Academy of Sciences of the United States of America 20130906 41

We have expanded the livestock gene editing toolbox to include transcription activator-like (TAL) effector nuclease (TALEN)- and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-stimulated homology-directed repair (HDR) using plasmid, rAAV, and oligonucleotide templates. Toward the genetic dehorning of dairy cattle, we introgressed a bovine POLLED allele into horned bull fibroblasts. Single nucleotide alterations or small indels were introduced into 14 additional genes in ...[more]

PMID: 24014591

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Project description:Soybean meal is a major component of livestock feed due to its high content and quality of protein. Understanding the genetic control of protein is essential to develop new cultivars with improved meal protein. Previously, a genomic region on chromosome 20 significantly associated with elevated protein content was identified in the cultivar Danbaekkong. The present research aimed to introgress the Danbaekkong high-protein allele into elite lines with different genetic backgrounds by developing and deploying robust DNA markers. A multiparent population consisting of 10 F5-derived populations with a total of 1,115 recombinant inbred lines (RILs) was developed using "Benning HP" as the donor parent of the Danbaekkong high-protein allele. A new functional marker targeting the 321-bp insertion in the gene Glyma.20g085100 was developed and used to track the Danbaekkong high-protein allele across the different populations and enable assessment of its effect and stability. Across all populations, the high-protein allele consistently increased the content, with an increase of 3.3% in seed protein. A total of 103 RILs were selected from the multiparent population for yield testing in five environments to assess the impact of the high-protein allele on yield and to enable the selection of new breeding lines with high protein and high yield. The results indicated that the high-protein allele impacts yield negatively in general; however, it is possible to select high-yielding lines with high protein content. An analysis of inheritance of the Chr 20 high-protein allele in Danbaekkong indicated that it originated from a Glycine soja line (PI 163453) and is the same as other G. soja lines studied. A survey of the distribution of the allele across 79 G. soja accessions and 35 Glycine max ancestors of North American soybean cultivars showed that the high-protein allele is present in all G. soja lines evaluated but not in any of the 35 North American soybean ancestors. These results demonstrate that G. soja accessions are a valuable source of favorable alleles for improvement of protein composition.

Dataset Information

Efficient nonmeiotic allele introgression in livestock using custom endonucleases.

Publications

Efficient nonmeiotic allele introgression in livestock using custom endonucleases.

Similar Datasets

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