Modeling human point mutation diseases in Xenopus tropicalis with a modified CRISPR/Cas9 system
Modeling human point mutation diseases in Xenopus tropicalis with a modified CRISPR/Cas9 system
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使用改良的 CRISPR/Cas9 系统对热带爪蟾的人类点突变疾病进行建模
DOI：
10.1096/fj.201802661r
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发表时间：
2019-06-01
期刊：
影响因子：
4.8
通讯作者：
Chen, Yonglong
中科院分区：
文献类型：
--
作者：
Shi, Zhaoying;Xin, Huhu;Chen, Yonglong
对热带非洲爪蟾进行精确的单碱基编辑将极大地扩展这种真正的二倍体青蛙在模拟由点突变引起的人类遗传疾病方面的效用。在这里，我们报道了利用大鼠载脂蛋白B mRNA编辑酶催化亚基1- xten聚集规则间隔短回文重复相关蛋白9 （Cas9）镍酶-尿嘧啶DNA糖基酶抑制剂-核定位序列碱基编辑器[碱基编辑器3 (BE3)]在热带棘猴中有效地转化C-to-T或G-to-A。将引导RNA和Cas9突变体复合体mRNA共注射到1细胞期热带天蛾胚胎中，在19个测试位点中有14个位点实现了精确的C-to-T或G-to-A替代，效率为5-75%，这使得稳定系的建立变得容易。BE3系统针对热带棘球蚴保守的T-box 5 R237和Tyr C28残基，分别模拟人类Holt-Oram综合征和1A型眼皮肤白化病。我们的数据表明，BE3是一个简单有效的工具，用于精确的碱基编辑。-施，忠，辛，海，田，东，莲，军，王，军，刘，国，冉，荣，施，生，张，志，施，艳，邓，艳，侯，昌，陈，艳。基于改进的CRISPR/Cas9系统建模热带爪蟾人类点突变疾病。
Precise single-base editing in Xenopus tropicalis would greatly expand the utility of this true diploid frog for modeling human genetic diseases caused by point mutations. Here, we report the efficient conversion of C-to-T or G-to-A in X. tropicalis using the rat apolipoprotein B mRNA editing enzyme catalytic subunit 1-XTEN-clustered regularly interspaced short palindromic repeat-associated protein 9 (Cas9) nickase-uracil DNA glycosylase inhibitor-nuclear localization sequence base editor [base editor 3 (BE3)]. Coinjection of guide RNA and the Cas9 mutant complex mRNA into 1-cell stage X. tropicalis embryos caused precise C-to-T or G-to-A substitution in 14 out of 19 tested sites with efficiencies of 5-75%, which allowed for easy establishment of stable lines. Targeting the conserved T-box 5 R237 and Tyr C28 residues in X. tropicalis with the BE3 system mimicked human Holt-Oram syndrome and oculocutaneous albinism type 1A, respectively. Our data indicate that BE3 is an easy and efficient tool for precise base editing in X. tropicalis.-Shi, Z., Xin, H., Tian, D., Lian, J., Wang, J., Liu, G., Ran, R., Shi, S., Zhang, Z., Shi, Y., Deng, Y., Hou, C., Chen, Y. Modeling human point mutation diseases in Xenopus tropicalis with a modified CRISPR/Cas9 system.