title: Highly Efficient Genome Editing via CRISPR/Cas9 to Create Clock Gene Knockout Cells.
abstract: Targeted genome editing using CRISPR/Cas9 is a relatively new, revolutionary technology allowing for efficient and directed alterations of the genome. It has been widely used for loss-of-function studies in animals and cell lines but has not yet been used to study circadian rhythms. Here, we describe the application of CRISPR/Cas9 genome editing for the generation of an F-box and leucine-rich repeat protein 3 (Fbxl3) knockout in a human cell line. Genomic alterations at the Fbxl3 locus occurred with very high efficiency (70%-100%) and specificity at both alleles, resulting in insertions and deletions that led to premature stop codons and hence FBXL3 knockout. Fbxl3 knockout cells displayed low amplitude and long period oscillations of Bmal1-luciferase reporter activity as well as increased CRY1 protein stability in line with previously published phenotypes for Fbxl3 knockout in mice. Thus, CRISPR/Cas9 genome editing should be highly valuable for studying circadian rhythms not only in human cells but also in classic model systems as well as nonmodel organisms.
authors.affiliation: Laboratory of Chronobiology, Charité Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Korge
authors.affiliation: Laboratory of Chronobiology, Charité Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Astrid
authors.initials: A
authors.lastname: Grudziecki
authors.affiliation: Laboratory of Chronobiology, Charité Universitätsmedizin Berlin, Berlin, Germany achim.kramer@charite.de.
authors.forename: Achim
authors.initials: A
authors.lastname: Kramer
mesh_terms: D056930:ARNTL Transcription Factors / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D064113:CRISPR-Cas Systems / Q000235:genetics*; D045744:Cell Line, Tumor; D057906:Circadian Clocks / Q000235:genetics* / Q000502:physiology; D056931:Cryptochromes / Q000235:genetics / Q000378:metabolism; D044783:F-Box Proteins / Q000235:genetics* / Q000378:metabolism; D055786:Gene Knockout Techniques / Q000379:methods*; D015894:Genome, Human / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008957:Models, Genetic; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D056930:ARNTL Transcription Factors; C538960:BMAL1 protein, human; C538963:CRY1 protein, human; D056931:Cryptochromes; D044783:F-Box Proteins; C519795:FBXL3 protein, human; D008156:Luciferases
keywords: CRISPR/Cas9; Fbxl3; circadian rhythms; clock genes; genome editing