Genome-Wide CRISPR/Cas9-Based Screening for Deubiquitinase Subfamily Identifies Ubiquitin-Specific Protease 11 as a Novel Regulator of Osteogenic Differentiation

Kamini Kaushal, Apoorvi Tyagi, Janardhan Keshav Karapurkar, Eun Jung Kim, Parthasaradhireddy Tanguturi, Kye Seong Kim, Han Sung Jung, Suresh Ramakrishna

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2 Citations (Scopus)


The osteoblast differentiation capacity of mesenchymal stem cells must be tightly regulated, as inadequate bone mineralization can lead to osteoporosis, and excess bone formation can cause the heterotopic ossification of soft tissues. The balanced protein level of Msh homeobox 1 (MSX1) is critical during normal osteogenesis. To understand the factors that prevent MSX1 protein degradation, the identification of deubiquitinating enzymes (DUBs) for MSX1 is essential. In this study, we performed loss-of-function-based screening for DUBs regulating MSX1 protein levels using the CRISPR/Cas9 system. We identified ubiquitin-specific protease 11 (USP11) as a protein regulator of MSX1 and further demonstrated that USP11 interacts and prevents MSX1 protein degradation by its deubiquitinating activity. Overexpression of USP11 enhanced the expression of several osteogenic transcriptional factors in human mesenchymal stem cells (hMSCs). Additionally, differentiation studies revealed reduced calcification and alkaline phosphatase activity in USP11-depleted cells, while overexpression of USP11 enhanced the differentiation potential of hMSCs. These results indicate the novel role of USP11 during osteogenic differentiation and suggest USP11 as a potential target for bone regeneration.

Original languageEnglish
Article number856
JournalInternational journal of molecular sciences
Issue number2
Publication statusPublished - 2022 Jan 1

Bibliographical note

Funding Information:
Author Contributions: K.K. and A.T. conducted all the experiments with the assistance of S.R., K.-S.K. and H.-S.J.; P.T. assisted screening experiments. J.K.K. and E.-J.K. assisted in differentiation experiments in hMSCs. The experimental strategy was designed by K.K., A.T. and S.R., K.K., A.T. and S.R. co-wrote the manuscript. K.-S.K., H.-S.J. and S.R. procured financial support. All authors have read and agreed to the published version of the manuscript.

Publisher Copyright:
© 2022 by the authors. Licensee MDPI, Basel, Switzerland.

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Molecular Biology
  • Spectroscopy
  • Computer Science Applications
  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Inorganic Chemistry


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