Efficient nonmeiotic allele introgression in livestock using custom endonucleases
Efficient nonmeiotic allele introgression in livestock using custom endonucleases
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DOI：
10.1073/pnas.1310478110
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发表时间：
2013-10-08
影响因子：
11.1
通讯作者：
Fahrenkrug, Scott C.
中科院分区：
文献类型：
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作者：
Tan, Wenfang;Carlson, Daniel F.;Fahrenkrug, Scott C.
我们扩展了家畜基因编辑工具箱，包括转录激活子样 (TAL) 效应核酸酶 (TALEN)，以及使用质粒、rAAV 和寡核苷酸模板的规则间隔短回文重复序列 (CRISPR)/Cas9 刺激的同源定向修复 (HDR)。为了实现奶牛的遗传去角，我们将牛 POLLED 等位基因渗入有角的公牛成纤维细胞中。使用 TALEN mRNA 和寡核苷酸转染将单核苷酸改变或小插入缺失引入猪、山羊和牛成纤维细胞中的 14 个附加基因，群体中的效率为 10-50%。一些选定的编辑模仿自然发生的性能增强或抗病等位基因，包括单碱基对的改变。高达 70% 未经选择而繁殖的成纤维细胞集落含有预期的编辑，其中一半以上是纯合的。编辑后的成纤维细胞被用来培育具有 DAZL 和 APC 基因敲除等位基因的猪，以模拟不育症和结肠癌。我们的方法能够实现牲畜中选定等位基因前所未有的无减数分裂种内和种间渗入，用于农业和生物医学应用。
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.