Exploring the functions of nonclassical MHC class Ib genes in Xenopus laevis by the CRISPR/Cas9 system.
Exploring the functions of nonclassical MHC class Ib genes in Xenopus laevis by the CRISPR/Cas9 system.
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DOI：
10.1016/j.ydbio.2016.05.023
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发表时间：
2017-06-15
影响因子：
2.7
通讯作者：
Robert J
中科院分区：
文献类型：
--
作者：
Banach M;Edholm ES;Robert J
非洲爪蟾非经典MHC类Ib （XNC）基因是一个高度相关和聚集的大家族，影响非洲爪蟾的免疫和潜在的其他生理功能。利用RNA干扰（RNAi）技术，我们先前证明了XNC基因之一XNC10.1对特异性先天T （iT）细胞群的发育和功能至关重要。然而，RNAi的限制，如F0和F1代中基因沉默的可变和不稳定程度，阻碍了对XNC10.1和其他XNC基因的彻底功能分析。为了克服这一障碍，我们对XNC基因采用了CRISPR/ cas9介导的基因编辑技术。我们高效、特异性地构建了XNC10.1、XNC11和XNC1的单基因敲除，以及XNC10.1和XNC11的双基因敲除。在单敲除XNC10.1的蝌蚪中，XNC10.1和Vα6-Jα1.43不变T细胞受体重排转录物的缺失表明XNC10.1在Vα6-Jα1.43 iT细胞中功能丧失和缺陷。值得注意的是，靶向XNC10.1不影响邻近的XNC基因，具有较高的序列相似性。此外，XNC1基因的破坏导致了发育阶段47的死亡，这表明该基因具有一些非免疫但必要的功能。这些数据表明，CRISPR/Cas9系统可以成功地用于F0代X射线的遗传分析。
A large family of highly related and clustered Xenopus nonclassical MHC class Ib (XNC) genes influences Xenopus laevis immunity and potentially other physiological functions. Using RNA interference (RNAi) technology, we previously demonstrated that one of XNC genes, XNC10.1, is critical for the development and function of a specialized innate T (iT) cell population. However, RNAi limitation such as a variable and unstable degree of gene silencing in F0 and F1 generations is hampering a thorough functional analysis of XNC10.1 and other XNC genes. To overcome this obstacle, we adapted the CRISPR/Cas9-mediated gene editing technique for XNC genes. We efficiently and specifically generated single gene knockouts of XNC10.1, XNC11, and XNC1 as well as double gene knockouts of XNC10.1 and XNC11 in X. laevis. In single XNC10.1 knockout X. laevis tadpoles, the absence of XNC10.1 and Vα6-Jα1.43 invariant T cell receptor rearrangement transcripts indicated XNC10.1 loss-of-function and deficiency in Vα6-Jα1.43 iT cells. Notably, targeting XNC10.1 did not affect neighboring XNC genes exhibiting high sequence similarity. Furthermore, XNC1 gene disruption induced mortality during developmental stage 47, suggesting some non-immune but essential function of this gene. These data demonstrate that the CRISPR/Cas9 system can be successfully adapted for genetic analysis in F0 generation of X laevis.
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DOI：
10.4049/jimmunol.1001467
发表时间：
2011-01-01
期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
--
作者：
Goyos A;Sowa J;Ohta Y;Robert J
通讯作者：
Robert J
影响因子：
1.5
作者：
Blitz, Ira L.;Biesinger, Jacob;Xie, Xiaohui;Cho, Ken W. Y.
通讯作者：
Cho, Ken W. Y.
影响因子：
14.9
作者：
Cradick TJ;Fine EJ;Antico CJ;Bao G
通讯作者：
Bao G
RNA/Cas9 介导的热带爪蟾基因组高效编辑
影响因子：
4.6
作者：
Guo, Xiaogang;Zhang, Tiejun;Chen, Yonglong
通讯作者：
Chen, Yonglong
影响因子：
5.4
作者：
Grayfer, Leon;Andino, Francisco De Jess;Robert, Jacques
通讯作者：
Robert, Jacques