An efficient miRNA knockout approach using CRISPR-Cas9 in Xenopus.
An efficient miRNA knockout approach using CRISPR-Cas9 in Xenopus.
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DOI：
10.1016/j.ydbio.2021.12.015
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发表时间：
2022-03
影响因子：
2.7
通讯作者：
Wheeler GN
中科院分区：
文献类型：
--
作者：
Godden AM;Antonaci M;Ward NJ;van der Lee M;Abu-Daya A;Guille M;Wheeler GN
近年来，CRISPR-Cas9敲除（KO）越来越多地用于研究基因功能。MicroRNAs （miRNAs）是一种短的非编码rna，长20-22个核苷酸，通过转录后抑制影响基因表达。我们之前发现miRNAs-196a和- 219与爪蟾神经嵴（NC）的发育有关。NC是一种多能干细胞群，在早期神经发育期间被指定。EMT后，NC细胞迁移到发育中的胚胎的各个点，在那里它们产生许多组织，包括部分周围神经系统、色素细胞和颅面骨骼。NC发育的失调导致了许多被归类为神经病变的疾病。由于mirna非常小，很难设计出可重复导致KO的CRISPR sgrna。因此，我们设计了一种新的方法，使用两种引导rna来有效地“删除”miRNA。我们敲除了miR-196a和miR-219，并将结果与相同mirna的morpholino敲低（KD）进行了比较。有效的CRISPR miRNA KO验证和表型分析包括使用关键NC和神经板边界标记（如Pax3， Xhe2， Sox10和Snail2）的全安装原位杂交，q-RT-PCR和Sanger测序。为了显示特异性，我们还使用miRNA模拟物挽救了敲除表型。MiRNA-219和miR-196a KO都显示NC缺失，神经板和孵化腺表型改变。蝌蚪显示大体颅面和色素表型。我们开发了一种新的CRISPR-Cas9方法来检测KO mirna。miR-196a的缺失导致色素表型的缺失。miR-219的缺失导致颅面表型。mirna可以影响爪蟾神经嵴的发育。
In recent years CRISPR-Cas9 knockouts (KO) have become increasingly ultilised to study gene function. MicroRNAs (miRNAs) are short non-coding RNAs, 20–22 nucleotides long, which affect gene expression through post-transcriptional repression. We previously identified miRNAs-196a and −219 as implicated in the development of Xenopus neural crest (NC). The NC is a multipotent stem-cell population, specified during early neurulation. Following EMT, NC cells migrate to various points in the developing embryo where they give rise to a number of tissues including parts of the peripheral nervous system, pigment cells and craniofacial skeleton. Dysregulation of NC development results in many diseases grouped under the term neurocristopathies. As miRNAs are so small, it is difficult to design CRISPR sgRNAs that reproducibly lead to a KO. We have therefore designed a novel approach using two guide RNAs to effectively ‘drop out’ a miRNA. We have knocked out miR-196a and miR-219 and compared the results to morpholino knockdowns (KD) of the same miRNAs. Validation of efficient CRISPR miRNA KO and phenotype analysis included use of whole-mount in situ hybridization of key NC and neural plate border markers such as Pax3, Xhe2, Sox10 and Snail2, q-RT-PCR and Sanger sequencing. To show specificity we have also rescued the knockout phenotype using miRNA mimics. MiRNA-219 and miR-196a KO’s both show loss of NC, altered neural plate and hatching gland phenotypes. Tadpoles show gross craniofacial and pigment phenotypes. We have developed a novel CRISPR-Cas9 approach to KO miRNAs. Loss of miR-196a leads to loss of pigment phenotypes. Loss of miR-219 leads to craniofacial phenotypes. MiRNAs can affect the development of Xenopus neural crest.
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影响因子：
4.6
作者：
Chang H;Yi B;Ma R;Zhang X;Zhao H;Xi Y
通讯作者：
Xi Y
影响因子：
2.7
作者：
Devotta A;Juraver-Geslin H;Gonzalez JA;Hong CS;Saint-Jeannet JP
通讯作者：
Saint-Jeannet JP
影响因子：
2.5
作者：
Dhorne-Pollet, Sophie;Thelie, Aurore;Pollet, Nicolas
通讯作者：
Pollet, Nicolas
影响因子：
28.1
作者：
Kim, Hui Kwon;Lee, Sungtae;Kim, Hyongbum Henry
通讯作者：
Kim, Hyongbum Henry
影响因子：
11.8
作者：
Chalmers, AD;Welchman, D;Papalopulu, N
通讯作者：
Papalopulu, N