Abstract
Many long noncoding RNAs (lncRNAs) have been identified through siRNA-based screening as essential regulators of embryonic stem cell (ESC) pluripotency. However, the biological and molecular functions of most lncRNAs remain unclear. Here, we employed CRISPR/Cas9-mediated knockout technology to explore the functions of 8 lncRNAs previously reported to promote pluripotency in mouse ESCs. Unexpectedly, all of these lncRNAs were dispensable for pluripotency maintenance and proliferation in mouse ESCs when disrupted individually or in combination. Single-cell transcriptomic analysis also showed that the knockout of these lncRNAs has a minimal impact on pluripotency gene expression and cell identity. We further showed that several small hairpin RNAs (shRNAs) previously used to knock down lncRNAs caused the downregulation of pluripotency genes in the corresponding lncRNA-knockout ESCs, indicating that off-target effects likely responsible for the pluripotency defects caused by these shRNAs. Interestingly, linc1343-knockout and linc1343-knockdown ESCs failed to form cystic structures and exhibited high expression of pluripotency genes during embryoid body (EB) differentiation. By reintroducing RNA products generated from the linc1343 locus, we found that two snoRNAs, Snora73a and Snora73b, but not lncRNAs, could rescue pluripotency silencing defects during EB differentiation of linc1343 knockout ESCs. Our results suggest that the 8 previously annotated pluripotency-regulating lncRNAs have no overt functions in conventional ESC culture; however, we identified snoRNA products derived from an annotated lncRNA locus as essential regulators for silencing pluripotency genes.
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Data availability
The accession numbers of the public and original datasets used in the study are included in Table S8 in Supporting Information. The dataset used in the present study is available in the NCBI GEO repository (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE225240).
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This work was supported by the National Natural Science Foundation of China (31900447, 32070792 and 91940302) and the Startup Foundation of Dermatology Hospital, Southern Medical University (2019RC06). The authors wish to thank the Bioinformatics Center at the Dermatology Hospital of Southern Medical University for providing high-performance computational resources.
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Li, Z., Li, X., Lin, J. et al. Reevaluation by the CRISPR/Cas9 knockout approach revealed that multiple pluripotency-associated lncRNAs are dispensable for pluripotency maintenance while Snora73a/b is essential for pluripotency exit. Sci. China Life Sci. 67, 2198–2212 (2024). https://doi.org/10.1007/s11427-023-2594-3
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DOI: https://doi.org/10.1007/s11427-023-2594-3