CRISPR-Cas9 is a powerful gene editing technique that can induce mutations in a target gene of interest in almost any mammalian cell line. However, its practicality can be limited if target cell lines are difficult to transfect and do not proliferate. In the current study, we have developed a streamlined approach for CRISPR-based gene knockouts with three key advantages, which allows phenotypic assay of gene knockouts without clonal selection and expansion. First, it integrates into a single, all-in-one vector transgenes for Cas9, sgRNA, and a fluorescence marker. Second, we used the Gateway system to rapidly clone specific sgRNAs into the all-in-one vector through PCR and in vitro recombination, without conventional enzyme digestion and ligation. Third, it uses adenovirus for the capacity to package the all-in-one vector, and for its high efficiency of transduction. We tested the all-in-one adenoviral CRISPR-Cas9 in a circadian clock model cell line U2OS, and demonstrated that essential clock genes such as Bmal1 and Per1 were knocked out so efficiently that functional assays could be performed from the heterogenic population without any clonal selection and expansion. This streamlined approach may prove invaluable for rapid functional assays of candidate genes in diverse biological pathways, including the circadian clock.