title: CRISPR/Cas9 allows efficient and complete knock-in of a destabilization domain-tagged essential protein in a human cell line, allowing rapid knockdown of protein function.
abstract: Although modulation of protein levels is an important tool for study of protein function, it is difficult or impossible to knockdown or knockout genes that are critical for cell growth or viability. For such genes, a conditional knockdown approach would be valuable. The FKBP protein-based destabilization domain (DD)-tagging approach, which confers instability to the tagged protein in the absence of the compound Shield-1, has been shown to provide rapid control of protein levels determined by Shield-1 concentration. Although a strategy to knock-in DD-tagged protein at the endogenous loci has been employed in certain parasite studies, partly due to the relative ease of knock-in as a result of their mostly haploid lifecycles, this strategy has not been demonstrated in diploid or hyperploid mammalian cells due to the relative difficulty of achieving complete knock-in in all alleles. The recent advent of CRISPR/Cas9 homing endonuclease-mediated targeted genome cleavage has been shown to allow highly efficient homologous recombination at the targeted locus. We therefore assessed the feasibility of using CRISPR/Cas9 to achieve complete knock-in to DD-tag the essential gene Treacher Collins-Franceschetti syndrome 1 (TCOF1) in human 293T cells. Using a double antibiotic selection strategy to select clones with at least two knock-in alleles, we obtained numerous complete knock-in clones within three weeks of initial transfection. DD-TCOF1 expression in the knock-in cells was Shield-1 concentration-dependent, and removal of Shield-1 resulted in destabilization of DD-TCOF1 over the course of hours. We further confirmed that the tagged TCOF1 retained the nucleolar localization of the wild-type untagged protein, and that destabilization of DD-TCOF1 resulted in impaired cell growth, as expected for a gene implicated in ribosome biogenesis. CRISPR/Cas9-mediated homologous recombination to completely knock-in a DD tag likely represents a generalizable and efficient strategy to achieve rapid modulation of protein levels in mammalian cells.
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Arnold
authors.initials: A
authors.lastname: Park
authors.affiliation: Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
authors.forename: Sohui T
authors.initials: ST
authors.lastname: Won
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Mickey
authors.initials: M
authors.lastname: Pentecost
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Wojciech
authors.initials: W
authors.lastname: Bartkowski
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, California, United States of America; Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
authors.forename: Benhur
authors.initials: B
authors.lastname: Lee
mesh_terms: D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D055879:Gene Knock-In Techniques / Q000379:methods*; D059765:Homologous Recombination; D006801:Humans; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics*; D010750:Phosphoproteins / Q000096:biosynthesis / Q000235:genetics*; D017434:Protein Structure, Tertiary
chemical_list: D009687:Nuclear Proteins; D010750:Phosphoproteins; C105442:TCOF1 protein, human
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