Generation of megabase-scale deletions, inversions and duplications involving the Contactin-6 gene in mice by CRISPR/Cas9 technology.
Generation of megabase-scale deletions, inversions and duplications involving the Contactin-6 gene in mice by CRISPR/Cas9 technology.
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DOI：
10.1186/s12863-017-0582-7
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发表时间：
2017-12-28
期刊：
影响因子：
2.9
通讯作者：
Serov OL
中科院分区：
文献类型：
--
作者：
Korablev AN;Serova IA;Serov OL
人类CNTN6基因(编码Contact-6蛋白)的拷贝数变异(CNV)由缺失或复制引起，导致严重的神经发育障碍，通常合并面部变形。相反，该基因的有害点突变不会表现出任何临床表型。本研究的目的是建立携带Cntn6基因大片段缺失、重复和倒置的小鼠，作为研究人类CNTN6基因座CNV的新的实验模型。为了在小鼠6号染色体上产生大的染色体重排，我们将CRISPR/Cas9技术应用于受精卵。将两个位于1137kb DNA片段两侧的引导RNA(GRNAs)与Cas9mRNA和单链DNA寡核苷酸(SsODN)一起显微注射到599个受精卵的细胞质中，并将其中256个移植到CD-1雌性小鼠的输卵管中。结果，我们观察到了41只存活的F0后代的出生。通过聚合酶链式反应分析和产物测序对这些小鼠进行基因分型。在41只F0后代中，我们发现了7只缺失的小鼠，2只携带重复基因的动物，4只携带倒置的小鼠。有趣的是，两个F0后代既有缺失又有复制。值得注意的是，虽然7个缺失载体中有3个在新的联合位点显示了预期的序列，但在另外3个缺失载体中，我们发现CRISPR/Cas9切割位点上缺少1-10个核苷酸，在一种动物中，103个核苷酸缺失，可能是由于容易出错的非同源末端连接。此外，我们在两个F0重复载体中检测到在这些位点上没有5个和13个核苷酸。在倒位的左右边界观察到CRISPR/Cas9酶切位点上相似的序列变化。因此，在分析的41个F0后代中，有11个发现了百万碱基规模的缺失、重复和倒置，即大约25%的效率。所有转基因的F0后代都是存活的，并能够将这些大的染色体重排传递给下一代。利用CRISPR/Cas9技术，我们创造了携带涉及全尺寸Cntn6基因的百万碱基规模的缺失、复制和倒置的小鼠。这些小鼠成为了新的小鼠品系的创建者，这可能是比Сntn6基因敲除小鼠更适合人类3p26.3区域CNV的实验模型。
Copy Number Variation (CNV) of the human CNTN6 gene (encoding the contactin-6 protein), caused by deletions or duplications, is responsible for severe neurodevelopmental impairments, often in combination with facial dysmorphias. Conversely, deleterious point mutations of this gene do not show any clinical phenotypes. The aim of this study is to generate mice carrying large deletions, duplications and inversions involving the Cntn6 gene as a new experimental model to study CNV of the human CNTN6 locus. To generate large chromosomal rearrangements on mouse chromosome 6, we applied CRISPR/Cas9 technology in zygotes. Two guide RNAs (gRNAs) (flanking a DNA fragment of 1137 Mb) together with Cas9 mRNA and single-stranded DNA oligonucleotides (ssODN) were microinjected into the cytoplasm of 599 zygotes of F1 (C57BL x CBA) mice, and 256 of them were transplanted into oviducts of CD-1 females. As a result, we observed the birth of 41 viable F0 offspring. Genotyping of these mice was performed by PCR analysis and sequencing of PCR products. Among the 41 F0 offspring, we identified seven mice with deletions, two animals carrying duplications of the gene and four carrying inversions. Interestingly, two F0 offspring had both deletions and duplications. It is important to note that while three of seven deletion carriers showed expected sequences at the new joint sites, in another three, we identified an absence of 1–10 nucleotides at the CRISPR/Cas9 cut sites, and in one animal, 103 bp were missing, presumably due to error-prone non-homologous end joining. In addition, we detected the absence of 5 and 13 nucleotides at these sites in two F0 duplication carriers. Similar sequence changes at CRISPR/Cas9 cut sites were observed at the right and left boundaries of inversions. Thus, megabase-scale deletions, duplications and inversions were identified in 11 F0 offspring among 41 analyzed, i.e., approximately 25% efficiency. All genetically modified F0 offspring were viable and able to transmit these large chromosomal rearrangements to the next generation. Using CRISPR/Cas9 technology, we created mice carrying megabase-scale deletions, duplications, and inversions involving the full-sized Cntn6 gene. These mice became founders of new mouse lines, which may be more appropriate experimental models of CNV in the human 3p26.3 region than Сntn6 knockout mice.
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