Inclusion of homologous DNA in nuclease-mediated gene targeting facilitates a higher incidence of bi-allelically modified cells
Inclusion of homologous DNA in nuclease-mediated gene targeting facilitates a higher incidence of bi-allelically modified cells
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DOI：
10.1111/xen.12194
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发表时间：
2015-09-01
影响因子：
3.9
通讯作者：
Prather, Randall S.
中科院分区：
文献类型：
--
作者：
Beaton, Benjamin P.;Kwon, Deug-Nam;Prather, Randall S.
背景：基因编辑技术的最新进展在数量和实用性上都有所增加。由于基因编辑的易用性和高效率，这些技术是通过同源重组来替代传统基因打靶方法的一种有吸引力的替代方法。我们之前已经在明尼苏达州的小型猪遗传背景中培育了胞苷一磷酸-N-乙酰神经氨酸羟基酶(CMAH)基因敲除(KO)猪。这些猪是用锌指核酸酶(ZFN)和供体DNA组合而成的，供体DNA的总同源性长度为1600bp(每条手臂上有800bp的同源性)。我们的下一个目标是介绍CMAHKO遗传背景中α-1，3-半乳糖基转移酶(GGTA1)的靶向性破坏，并评价供体DNA同源长度对巨核酸酶介导的基因靶向的影响。方法：使用先前CMAHKO实验中的锌指核酸酶作为概念证明，确定供体DNA同源长度与靶向效率之间的相关性。在这些结果的基础上，设计了使用转录激活物样效应核酸酶(TALEN)来产生双等位修饰的GGTA1细胞的实验，该细胞使用带有不同长度同源性的供体DNA。供体DNA被设计成对称地位于CMAH和GGTA1的预测切割位点的两侧，分别针对ZFN和TALEN切割位点。两个基因的总同源性长度在60~1799个碱基之间。结果：将ZFN导入供体DNA后，在ZFN介导的CMAH靶向修复过程中，少量的同源DNA(60bp)可以促进同源定向修复；但同源性较高的供体DNA导致同源定向修复的频率较高。对于使用TALENS和供体DNA的GGTA1 KO实验，仅有供体DNA不会导致可检测到的GGTA1双等位基因转换。随着供体DNA长度的增加，GGTA1的双等位基因断裂率从0.5%(仅TALEN，不存在供体DNA)增加到最大3%(TALENS和供体DNA，总同源性为1799bp)。在TALEN介导的基因打靶中包含同源供体DNA有助于提高双等位基因修饰细胞的发生率。利用所产生的细胞，我们能够证明GGTA1的表达缺失，唾液酸转移酶相关基因的表达降低。结论：供体DNA与巨核酸酶联合使用的方法可以提高基因打靶的效率。这种基因编辑方法不仅适用于其他哺乳动物系统，也适用于其他基因。此外，基因表达分析进一步证实了CMAH/GGTA1双KO猪可以作为猪到人异种移植研究的有价值的来源。
Background: Recent advancements in gene editing techniques have increased in number and utility. These techniques are an attractive alternative to conventional gene targeting methods via homologous recombination due to the ease of use and the high efficiency of gene editing. We have previously produced cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) knockout (KO) pigs in a Minnesota miniature pig genetic background. These pigs were generated using zinc-finger nucleases (ZFNs) in combination with donor DNA containing a total homology length of 1600 bp (800-bp homology on each arm). Our next aim was to introduce the targeted disruption of alpha-1,3-galactosyltransferase (GGTA1) in the CMAH KO genetic background and evaluate the effect of donor DNA homology length on meganuclease-mediated gene targeting.Methods: Zinc-finger nucleases from a previous CMAH KO experiment were used as a proof of concept to identify a correlation between the length of donor DNA homology and targeting efficiency. Based on those results, experiments were designed to use transcription activator-like effector nucleases (TALENs) to generate bi-allelically modified GGTA1 cells using donor DNAs carrying various lengths of homology. Donor DNA was designed to symmetrically flank the predicted cleavage sites in CMAH and GGTA1 for both ZFN and TALEN cleavage sites, respectively. For both genes, the length of total homology ranged from 60 to 1799 bp. Sialyltransferase gene expression profiles were evaluated in CMAH and GGTA1 double KO pig cells and were compared to wild-type and CMAH KO cells.Results: Introduction of donor DNA with ZFNs demonstrated that small amounts of homology (60 bp) could facilitate homology-directed repair during ZFN-mediated targeting of CMAH; however, donor DNA with longer amounts of homology resulted in a higher frequency of homology-directed repair. For the GGTA1 KO experiments that used TALENs and donor DNA, donor DNA alone did not result in detectable bi-allelic conversion of GGTA1. As the length of donor DNA increased, the bi-allelic disruption of GGTA1 increased from 0.5% (TALENs alone, no donor DNA present) to a maximum of 3% (TALENs and donor DNA with total homology of 1799 bp). Inclusion of homologous donor DNA in TALEN-mediated gene targeting facilitated a higher incidence of bi-allelically modified cells. Using the generated cells, we were able to demonstrate the lack of GGTA1 expression and the decrease in gene expression sialyltransferase-related genes.Conclusions: The approach of using donor DNA in conjunction with a meganuclease can be used to increase the efficiency of gene targeting. The gene editing methods can be applied to other genes as well as other mammalian systems. Additionally, gene expression analysis further confirms that the CMAH/GGTA1 double KO pigs can be a valuable source for the study of pig-to-human xenotransplantation.