CRISPR-Cas9-mediated genome editing in one blastomere of two-cell embryos reveals a novel Tet3 function in regulating neocortical development

Wang, Lingbo; Li, Min Yin; Qu, Chao; Miao, Wan Ying; Yin, Qi; Liao, Jiaoyang; Cao, Hua; Huang, Min; Wang, Kai; Zuo, Erwei; Peng, Guangdun; Zhang, Shu Xin; Chen, Guodong; Li, Qing; Tang, Ke; Qian, Yu; Li, Zhoujie; Wong, Catherine C. L.; Xu, Guoliang; Jing, Naihe; Yu, Xiang; Li, Jinsong

doi:10.1038/cr.2017.58

Cited by 39 publications

(20 citation statements)

References 43 publications

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“…This is in line with our observation that Tet3 KD NPCs form clusters of cells that resemble ES-colonies and are OCT4-positive. Additionally, TET3 has been implicated in regulation of synaptic transmission [60,61] and fear-extinction memory [21], which suggests a pivotal role in the nervous system.…”

Section: Discussionmentioning

confidence: 99%

Tet3 regulates cellular identity and DNA methylation in neural progenitor cells

Santiago

Antunes

Guedes

et al. 2019

Cell. Mol. Life Sci.

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TET enzymes oxidize 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), a process thought to be intermediary in an active DNA demethylation mechanism. Notably, 5hmC is highly abundant in the brain and in neuronal cells. Here, we interrogated the function of Tet3 in neural precursor cells (NPCs), using a stable and inducible knockdown system and an in vitro neural differentiation protocol. We show that Tet3 is upregulated during neural differentiation, whereas Tet1 is downregulated. Surprisingly, Tet3 knockdown led to a de-repression of pluripotency-associated genes such as Oct4, Nanog or Tcl1, with concomitant hypomethylation. Moreover, in Tet3 knockdown NPCs, we observed the appearance of OCT4positive cells forming cellular aggregates, suggesting de-differentiation of the cells. Notably, Tet3 KD led to a genome-scale loss of DNA methylation and hypermethylation of a smaller number of CpGs that are located at neurogenesis-related genes and at imprinting control regions (ICRs) of Peg10, Zrsr1 and Mcts2 imprinted genes. Overall, our results suggest that TET3 is necessary to maintain silencing of pluripotency genes and consequently neural stem cell identity, possibly through regulation of DNA methylation levels in neural precursor cells.

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Section: Discussionmentioning

confidence: 99%

Tet3 regulates cellular identity and DNA methylation in neural progenitor cells

Santiago

Antunes

Guedes

et al. 2019

Cell. Mol. Life Sci.

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“…This ensures that organs will comprise cells of mixed progeny (wild type or mutant) derived from either of the two initial cells. The latter approach proved successful in a recent report where Tet3 functions were illustrated in the mosaic mutant (Wang et al, 2017). Although this is still an on-going study, we can obtain Atoh1 −/− mosaic mice that survive until at least postnatal day 7 (P7).…”

Section: Discussionmentioning

confidence: 95%

Simultaneous zygotic inactivation of multiple genes in mouse through CRISPR/Cas9-mediated base editing

et al. 2018

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In vivo genetic mutation has become a powerful tool for dissecting gene function; however, multi-gene interaction and the compensatory mechanisms involved can make findings from single mutations, at best difficult to interpret, and, at worst, misleading. Hence, it is necessary to establish an efficient way to disrupt multiple genes simultaneously. CRISPR/Cas9-mediated base editing disrupts gene function by converting a protein-coding sequence into a stop codon; this is referred to as CRISPR-stop. Its application in generating zygotic mutations has not been well explored yet. Here, we first performed a proof-of-principle test by disrupting Atoh1, a gene crucial for auditory hair cell generation. Next, we individually mutated vGlut3 (Slc17a8), otoferlin (Otof) and prestin (Slc26a5), three genes needed for normal hearing function. Finally, we successfully disrupted vGlut3, Otof and prestin simultaneously. Our results show that CRISPR-stop can efficiently generate single or triple homozygous F0 mouse mutants, bypassing laborious mouse breeding. We believe that CRISPR-stop is a powerful method that will pave the way for highthroughput screening of mouse developmental and functional genes, matching the efficiency of methods available for model organisms such as Drosophila.

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“…Fortunately, the F0-7 male rabbits could live to sexual maturity and were used for mating with the WT rabbits. The mosaicism in the F0-7 rabbits indicated that germline and genotype chimeras generated by CRISPR/Cas9 could significantly expand studies of unknown gene functions in cells or animals ( Wang et al, 2017 ). We could not obtain homozygous gene knockout rabbits owing to the high mortality rate of the FBN1 Het rabbits.…”

Section: Discussionmentioning

confidence: 99%

Truncated C-terminus of fibrillin-1 induces Marfanoid-progeroid-lipodystrophy (MPL) syndrome in rabbit

Chen

Yao

Yang

et al. 2018

Disease Models &Amp; Mechanisms

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Various clinical differences have been observed between patients with the FBN1 gene mutation and those with the classical Marfan phenotype. Although FBN1 knockout (KO) or dominant-negative mutant mice are widely used as an animal model for Marfan syndrome (MFS), these mice cannot recapitulate the genotype/phenotype relationship of Marfanoid-progeroid-lipodystrophy (MPL) syndrome, which is caused by a mutation in the C-terminus of fibrillin-1, the penultimate exon of the FBN1 gene. Here, we describe the generation of a rabbit MPL model with C-terminal truncation of fibrillin-1 using a CRISPR/Cas9 system. FBN1 heterozygous (FBN1 Het) rabbits faithfully recapitulated the phenotypes of MFS, including muscle wasting and impaired connective tissue, ocular syndrome and aortic dilation. Moreover, skin symptoms, lipodystrophy, growth retardation and dysglycemia were also seen in these FBN1 Het rabbits, and have not been reported in other animal models. In conclusion, this novel rabbit model mimics the histopathological changes and functional defects of MPL syndrome, and could become a valuable model for studies of pathogenesis and drug screening for MPL syndrome.

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CRISPR-Cas9-mediated genome editing in one blastomere of two-cell embryos reveals a novel Tet3 function in regulating neocortical development

Cited by 39 publications

References 43 publications

Tet3 regulates cellular identity and DNA methylation in neural progenitor cells

Tet3 regulates cellular identity and DNA methylation in neural progenitor cells

Simultaneous zygotic inactivation of multiple genes in mouse through CRISPR/Cas9-mediated base editing

Truncated C-terminus of fibrillin-1 induces Marfanoid-progeroid-lipodystrophy (MPL) syndrome in rabbit

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