Engineered Cas9 variants bypass Keap1-mediated degradation in human cells and enhance epigenome editing efficiency.

Autor:	Chen J; Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.; Department of Biochemistry and Biophysics, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA., Su S; Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.; Department of Biochemistry and Biophysics, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA., Pickar-Oliver A; Department of Biomedical Engineering, Duke University, Durham, NC 27710, USA.; Center for Advanced Genomic Technologies, Duke University, Durham, NC 27710, USA., Chiarella AM; 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.; Curriculum in Genetics and Molecular Biology, The University of North Carolina, Chapel Hill, NC 27599, USA., Hahn Q; Department of Biochemistry and Biophysics, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA., Goldfarb D; Department of Cell Biology and Physiology, Washington University, St. Louis, MO 63110, USA.; Institute for Informatics, Data Science & Biostatistics, Washington University, St. Louis, MO 63110, USA., Cloer EW; Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.; Department of Cell Biology and Physiology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA., Small GW; Center for Pharmacogenomics and Individualized Therapy, Eshelman School of Pharmacy, The University of North Carolina, Chapel Hill, NC 27599, USA., Sivashankar S; Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA., Ramsden DA; Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.; Department of Biochemistry and Biophysics, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA., Major MB; Department of Cell Biology and Physiology, 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.; 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.; Curriculum in Genetics and Molecular Biology, The University of North Carolina, Chapel Hill, NC 27599, USA., Gersbach CA; Department of Biomedical Engineering, Duke University, Durham, NC 27710, USA.; Center for Advanced Genomic Technologies, Duke University, Durham, NC 27710, USA., Liu P; Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.; Department of Biochemistry and Biophysics, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Jazyk:	angličtina
Zdroj:	Nucleic acids research [Nucleic Acids Res] 2024 Oct 28; Vol. 52 (19), pp. 11536-11551.
DOI:	10.1093/nar/gkae761
Abstrakt:	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. (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)
Databáze:	MEDLINE
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