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.

Linking Core Promoter Classes to Circadian Transcription

“…By targeting the core promoter, e.g. the TATA box, by gene editing techniques [ 73 , 74 ], it might be possible to more precisely attenuate rhythmicity while keeping average levels constant.…”

Adenosine A3 receptor elicits chemoresistance mediated by multiple resistance-associated protein-1 in human glioblastoma stem-like cells

“…For the generation of a human U87MG A 3 AR Knockout (U87MG KO ) cells we used the CRISPR-Cas9 system [48]. The plasmidial vector contained the Cas9 endonuclease and gRNA was purchased at Santa Cruz Biotechnology (sc-402007).…”

Single adeno-associated virus-based multiplexed CRISPR-Cas9 system to nullify core components of the mammalian molecular clock

“…In this study, we devised CSAC, a system of multiplexed sgRNAs targeting core clock components packaged in a single AAV, to abolish molecular clock oscillation by inducing null mutations into several core clock genes. We selected the multiplexed sgRNAs based on leucine-rich repeat protein 3 (Fbxl3) [23] . To do so, they transfected U2-OS cells stably expressing a Bmal1-Luciferase reporter with a lentivirus expressing Cas9 and two sgRNA targeting different sites in the Fbxl3 gene, and found that CRISPR-Cas9-based Fbxl3 KO dampened Bmal1 promoter-driven luminescence oscillation.…”

“…In circadian genes, several applications of the CRISPR-Cas9 system has been reported to KO molecular clock components in a robust and efficient manner. Korge et al first utilized CRISPR-Cas9mediated gene targeting in chronobiology to generate FBXL3 KO U2-OS cells [23] and have recently extended this application to generate CRY1 and CRY2 KO U2-OS cells. The CRISPR-Cas9 system has also been used to generate KO animals lacking core clock genes in mice, macaques, monarch butterflies, and Neurospora crassa [24][25][26][27] .…”