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Reevaluation by CRISPR-Cas9 approach reveals multiple lincRNAs dispensable for pluripotency maintenance and SNORA73 essential for pluripotency exit

ABSTRACT: This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Mus musculus

PROVIDER: GSE225240 | GEO | 2024/08/07

REPOSITORIES: GEO

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Reevaluation by CRISPR-Cas9 approach reveals multiple lincRNAs dispensable for pluripotency maintenance and SNORA73 essential for pluripotency exit (scRNA-Seq)

Project description:Many lincRNAs have been identified as essential regulators for ESC pluripotency through siRNA-based screening. However, the biological and molecular functions of the large intergenic noncoding RNAs (lincRNAs) remain unclear. Here, we employed CRISPR/Cas9-mediated knockout technology to explore the functions of 8 previously annotated lincRNAs in ESCs. Unexpectedly, it was found that most lincRNAs are dispensable for pluripotency maintenance, cloning formation ability, proliferation and apoptosis in ESCs when we disrupted these lincRNAs individually or in combinations. Single-cell transcriptomic analysis of a set of lncRNA knockout ESCs showed consistent results, indicating that previously observed functions of lincRNAs on ESC pluripotency maintenance may be due to off-targeted effects. Interestingly, deletion and transcriptional repression of linc1343 disrupts embryoid body (EB) differentiation in ESC. Notably, GM17300 and two small RNAs (Snora73a, Snora73b) derived from the lnc1343 locus are highly expressed in ESCs. Further overexpression of Snora73a and Snora73b in linc1343 knockout cells rescue the phenotype, suggesting that snoRNAs derived from linc1343 are the responders for EB differentiation. Taken together, our results demonstrate the divergent effects of lincRNAs on regulating ESC pluripotency and EB differentiation.

2024-08-07 | GSE225238 | GEO

Reevaluation by CRISPR-Cas9 approach reveals multiple lincRNAs dispensable for pluripotency maintenance and SNORA73 essential for pluripotency exit (RNA-Seq)

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Reevaluation by CRISPR-Cas9 approach reveals multiple lincRNAs dispensable for pluripotency maintenance and SNORA73 essential for pluripotency exit

Project description:Reevaluation by CRISPR-Cas9 approach reveals multiple lincRNAs dispensable for pluripotency maintenance and SNORA73 essential for pluripotency exit

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Reevaluation by CRISPR-Cas9 approach reveals multiple lincRNAs dispensable for pluripotency maintenance and SNORA73 essential for pluripotency exit (scRNA-Seq)

Project description:Reevaluation by CRISPR-Cas9 approach reveals multiple lincRNAs dispensable for pluripotency maintenance and SNORA73 essential for pluripotency exit (scRNA-Seq)

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Reevaluation by CRISPR-Cas9 approach reveals multiple lincRNAs dispensable for pluripotency maintenance and SNORA73 essential for pluripotency exit (RNA-Seq)

Project description:Reevaluation by CRISPR-Cas9 approach reveals multiple lincRNAs dispensable for pluripotency maintenance and SNORA73 essential for pluripotency exit (RNA-Seq)

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Screening Genes Promoting Exit from Naive Pluripotency Based on Genome-Scale CRISPR-Cas9 Knockout.

Project description:Two of the main problems of stem cell and regenerative medicine are the exit of pluripotency and differentiation to functional cells or tissues. The answer to these two problems holds great value in the clinical translation of stem cell as well as regenerative medicine research. Although piling researches have revealed the truth about pluripotency maintenance, the mechanisms underlying pluripotent cell self-renewal, proliferation, and differentiation into specific cell lineages or tissues are yet to be defined. To this end, we took full advantage of a novel technology, namely, the genome-scale CRISPR-Cas9 knockout (GeCKO). As an effective way of introducing targeted loss-of-function mutations at specific sites in the genome, GeCKO is able to screen in an unbiased manner for key genes that promote exit from pluripotency in mouse embryonic stem cells (mESCs) for the first time. In this study, we successfully established a model based on GeCKO to screen the key genes in pluripotency withdrawal. Our strategies included lentiviral package and infection technology, lenti-Cas9 gene knockout technology, shRNA gene knockdown technology, next-generation sequencing, model-based analysis of genome-scale CRISPR-Cas9 knockout (MAGeCK analysis), GO analysis, and other methods. Our findings provide a novel approach for large-scale screening of genes involved in pluripotency exit and offer an entry point for cell fate regulation research.

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Polycomb repressive complex 2 is dispensable for maintenance of embryonic stem cell pluripotency.

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CRISPR/Cas9-Mediated Rapid Generation of Multiple Mouse Lines Identified Ccdc63 as Essential for Spermiogenesis.

Project description:Spermatozoa are flagellated cells whose role in fertilization is dependent on their ability to move towards an oocyte. The structure of the sperm flagella is highly conserved across species, and much of what is known about this structure is derived from studies utilizing animal models. One group of proteins essential for the movement of the flagella are the dyneins. Using the advanced technology of CRISPR/Cas9 we have targeted three dynein group members; Dnaic1, Wdr63 and Ccdc63 in mice. All three of these genes are expressed strongly in the testis. We generated mice with amino acid substitutions in Dnaic1 to analyze two specific phosphorylation events at S124 and S127, and generated simple knockouts of Wdr63 and Ccdc63. We found that the targeted phosphorylation sites in Dnaic1 were not essential for male fertility. Similarly, Wdr63 was not essential for male fertility; however, Ccdc63 removal resulted in sterile male mice due to shortened flagella. This study demonstrates the versatility of the CRISPR/Cas9 system to generate animal models of a highly complex system by introducing point mutations and simple knockouts in a fast and efficient manner.

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lincRNAs act in the circuitry controlling pluripotency and differentiation.

Project description:Although thousands of large intergenic non-coding RNAs (lincRNAs) have been identified in mammals, few have been functionally characterized, leading to debate about their biological role. To address this, we performed loss-of-function studies on most lincRNAs expressed in mouse embryonic stem (ES) cells and characterized the effects on gene expression. Here we show that knockdown of lincRNAs has major consequences on gene expression patterns, comparable to knockdown of well-known ES cell regulators. Notably, lincRNAs primarily affect gene expression in trans. Knockdown of dozens of lincRNAs causes either exit from the pluripotent state or upregulation of lineage commitment programs. We integrate lincRNAs into the molecular circuitry of ES cells and show that lincRNA genes are regulated by key transcription factors and that lincRNA transcripts bind to multiple chromatin regulatory proteins to affect shared gene expression programs. Together, the results demonstrate that lincRNAs have key roles in the circuitry controlling ES cell state.

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Project description: Not available

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