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Engineered Cas9 variants bypass Keap1-mediated degradation in human cells and enhance epigenome editing efficiency.
Chen, Jianfeng; Su, Siyuan; Pickar-Oliver, Adrian; Chiarella, Anna M; Hahn, Quentin; Goldfarb, Dennis; Cloer, Erica W; Small, George W; Sivashankar, Smaran; Ramsden, Dale A; Major, Michael B; Hathaway, Nathaniel A; Gersbach, Charles A; Liu, Pengda.
Afiliação
  • Chen J; Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
  • Su S; Department of Biochemistry and Biophysics, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
  • Pickar-Oliver A; Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
  • Chiarella AM; Department of Biochemistry and Biophysics, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
  • Hahn Q; Department of Biomedical Engineering, Duke University, Durham, NC 27710, USA.
  • Goldfarb D; Center for Advanced Genomic Technologies, Duke University, Durham, NC 27710, USA.
  • Cloer EW; Division of Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, Eshelman School of Pharmacy, The University of North Carolina, Chapel Hill, NC 27599, USA.
  • Small GW; Curriculum in Genetics and Molecular Biology, The University of North Carolina, Chapel Hill, NC 27599, USA.
  • Sivashankar S; Department of Biochemistry and Biophysics, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
  • Ramsden DA; Department of Cell Biology and Physiology, Washington University, St. Louis, MO 63110, USA.
  • Major MB; Institute for Informatics, Data Science & Biostatistics, Washington University, St. Louis, MO 63110, USA.
  • Hathaway NA; Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
  • Gersbach CA; Department of Cell Biology and Physiology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
  • Liu P; Center for Pharmacogenomics and Individualized Therapy, Eshelman School of Pharmacy, The University of North Carolina, Chapel Hill, NC 27599, USA.
Nucleic Acids Res ; 2024 Sep 04.
Article em En | MEDLINE | ID: mdl-39228373
ABSTRACT
As a potent and convenient genome-editing tool, Cas9 has been widely used in biomedical research and evaluated in treating human diseases. Numerous engineered variants of Cas9, dCas9 and other related prokaryotic endonucleases have been identified. However, as these bacterial enzymes are not naturally present in mammalian cells, whether and how bacterial Cas9 proteins are recognized and regulated by mammalian hosts remain poorly understood. Here, we identify Keap1 as a mammalian endogenous E3 ligase that targets Cas9/dCas9/Fanzor for ubiquitination and degradation in an 'ETGE'-like degron-dependent manner. Cas9-'ETGE'-like degron mutants evading Keap1 recognition display enhanced gene editing ability in cells. dCas9-'ETGE'-like degron mutants exert extended protein half-life and protein retention on chromatin, leading to improved CRISPRa and CRISPRi efficacy. Moreover, Cas9 binding to Keap1 also impairs Keap1 function by competing with Keap1 substrates or binding partners for Keap1 binding, while engineered Cas9 mutants show less perturbation of Keap1 biology. Thus, our study reveals a mammalian specific Cas9 regulation and provides new Cas9 designs not only with enhanced gene regulatory capacity but also with minimal effects on disrupting endogenous Keap1 signaling.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Idioma: En Revista: Nucleic Acids Res Ano de publicação: 2024 Tipo de documento: Article País de afiliação: Estados Unidos País de publicação: Reino Unido
Texto completo
Adicionar na Minha BVS
Imprimir
XML
PubMed Links
Buscar no Google

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Idioma: En Revista: Nucleic Acids Res Ano de publicação: 2024 Tipo de documento: Article País de afiliação: Estados Unidos País de publicação: Reino Unido