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

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

  • Stem Cells Int. 2020 Feb 3;2020:8483035. doi: 10.1155/2020/8483035.
Bin Yang  # 1 2 Junqi Kuang  # 3 Chuman Wu  # 3 Wenyi Zhou  # 2 Shuoji Zhu  # 2 Haodong Jiang 2 4 Ziwei Zhai 3 Yue Wu 4 Junwei Peng 4 Nanbo Liu 2 Haiyan Hu 1 2 Nasser Moussa Ide 2 Ruiping Chen 5 Mingyi Zhao 6 Ping Zhu 1 2
Affiliations

Affiliations

  • 1 The Second School of Clinical Medicine, Southern Medical University, Guangzhou, Guangdong 510515, China.
  • 2 Guangdong Institute of Cardiovascular Disease, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong 510080, China.
  • 3 Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong 510530, China.
  • 4 School of Medicine, South China University of Technology, Guangzhou, Guangdong 510006, China.
  • 5 Department of Cardiothoracic Surgery of East Division, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510080, China.
  • 6 Department of Pediatrics, The Third Xiangya Hospital, Central South University, Changsha, Hunan 410013, China.
  • # Contributed equally.
Abstract

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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