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A system for generating reversible knockout cell lines

A powerful use of gene editing technology is the generation of genetic-null (knockout) mutants of human immortalized cell lines. However, a problem that arises in the process of generating such edited lines is that to ensure edit homogeneity, clonal selection must often be performed and, given the genomic instability of immortalized cell lines and the high division number inherent to the clonal selection process, the resulting clone can exhibit genotypic and phenotypic differences from the parental line. Here, we present a system that allows for the generation of genetic null cell lines with an excisable cassette. This system allows for the generation of suitable controls without the need of further rounds of clonal selection.

as desired, its efficiency is low and, as with indel-based editing, clonal identification and selection must be carried out.
A key hurdle for both indel-mediated and HDR-mediated face relates to the lack of proper wild-type or unmodified controls. Frequently, what is used as a control is either the parental line, clones obtained from a "mock" modification process, or a combination of the two. Yet, the genomic instability of immortalized lines leads to significant drift in the characteristics of the resulting clones compared with the parental line (Barnes et al., 2006;Tharmalingam et al., 2018).
We have established a novel gene editing approach that allows us to disrupt gene expression and that can be reversed in an expanded clone population without the need for further selection -thus generating as ideal a control as currently technically possible.
In our approach, we use homologous recombination to split an exon and introduce a selectable and excisable disruption cassette that terminates both transcription and translation ( Figure. 1). Following selection of edited cells, the disruption cassette can then be excised by the action of Cre recombinase, rescuing gene expression.
As proof of principle, we applied our system to abrogate expression of the key circadian clock component BMAL1 (ARNTL) in U2OS cells given that Bmal1 loss in this line has been shown to have robust phenotypes (Baggs et al., 2009;Vollmers et al., 2008). First, we introduced a cassette into exon 8 of the Bmal1 gene, which codes for the basic Helix-Loop-Helix DNA-binding domain of BMAL1 and whose targeting has been used to generate genetic-null animals (Storch et al., 2007). Following antibiotic selection, single cells were plated in 96-well plates and three clonal lines were identified via PCR genotyping as both harboring the modified allele and lacking an unmodified one ( Figure   2). We observed loss of BMAL1 expression, and the expected associated loss of circadian rhythmicity as assessed by real-time luminescence measurements of a Bmal1 promoter-driven Luciferase reporter construct. We were able to rescue both BMAL1 expression and circadian rhythmicity by expression of Cre recombinase, which we delivered into the expanded clone population via an adenovirus vector (Figure 3). Of note, even though rhythmicity is restored in cells where BMAL1 expression has been rescued, the circadian profile is not identical to the original cell line (Figure 4), which is consistent with the aforementioned changes that occur during the clone selection process.
Transfections were performed using Trans-IT LT1 (TLT-1) (Mirus Bio) according to the manufacturer's instruction. To excise the disruption cassette, we seeded 2 million cells of the desired clone onto a 10 cm dish and delivered Cre via adenovirus (10 x10 6 pfu per 2 million cells, Vector BioLabs).
Real-time luciferase assays were performed in a 96-well plate format by reverse transfecting cells with 300 ng Bmal1-Luciferase reporter construct and measuring luminescence in a temperature-controlled microplate reader for up to 7 days.
Immunoblotting was performed with an Odyssey Imaging System (LiCor) and related reagents. Total protein content was visualized with Revert reagent (LiCor).

Oligonucleotides
All oligonucleotides were ordered from IDT.