Despite the advancements of genetic tools such as CRISPR/Cas9-based gene editing1, gene silencing2, or inducible gene expression approaches3, none of these systems displays readiness of activity compatible with the kinetics of cell cycle progression. Conversely, an alternative to obtain rapid degradation of the POI is represented by targeted proteolysis using polypeptide tags, also known as degrons4,5. In one of the most commonly used degron systems, the POI is fused with an Auxin-Inducible Degron (AID) sequence, such as the 7 kDa degron termed mini-AID (mAID), in cell lines expressing the Oryza sativa TIR1 (OsTIR1) F-box protein6,7. When the phytohormone auxin is provided, OsTIR1 binds the mAID-POI and induces its quick proteasomal degradation6,7. However, despite their speed, reversibility, and fine-tuning, degron-based systems still lack cell cycle phase-specificity and require conventional cell synchronization8, routinely achieved by exposing cells to stress conditions9,10.
Here, we report the engineering of the “Regulated OsTIR1 Levels of Expression based on the Cell-Cycle Status” (ROLECCS) technology, which combines the AID and the FUCCI systems. In this new tool, the OsTIR1 protein is fused to the fluorescent indicator mEmerald and the FUCCI11 tags Cdt1/Geminin, which are responsible for the restricted G1 and S/G2 expression, respectively. Upon auxin treatment, only the cells expressing the fusion-protein OsTIR1-mEmerald-Cdt1/Geminin, (i.e. in the desired cell cycle phase) degrade the mAID-POI.
In our design (Figure 1A-C, Supplementary Figure 1A), the OsTIR1 coding gene was fused in-frame with a mEmerald fluorescent reporter (a brightly fluorescent monomeric variant of GFP)12 that allows the identification of cells expressing these constructs. Then, we added the sequences corresponding to either human Cdt1 (aa 30-120) or Geminin (aa 1-110) to restrict OsTIR1 expression to different phases of the cell cycle, like in the FUCCI system. For convenience, the hCdt1 (30-120) and hGeminin (1-110) tags are indicated hereafter as Cdt1 and GEM, respectively. We also generated a construct where no additional tag was added, to allow OsTIR1-mEmerald expression independently on the phase of the cell cycle.
Engineered variants of OsTIR1-mEmerald, OsTIR1-mEmerald-Cdt1, and OsTIR1-mEmerald-GEM genes are actively transcribed throughout the cell cycle. However, the presence of the Cdt1 and the Geminin tags determine the Regulated OsTIR1 Levels of Expression based on the Cell Cycle Status (ROLECCS system). We predicted that the OsTIR1-mEmerald protein would be stably present throughout the cell cycle. Therefore, auxin treatment would trigger OsTIR1 enzymatic activity and degradation of the mAID-tagged protein of interest in any cell, independent of the cell cycle status (from now on: asynchronous ROLECCS, ROLECCS AS) (Figure 1A).
On the other hand, the presence of OsTIR1-mEmerald-Cdt1 (from now on: ROLECCS G1) protein would be restricted the G1/early S phase, because ubiquitylation by SCFSkp2 E3 ligase leads to its prompt degradation during S-phase transition. Thus, addition of auxin would lead to OsTIR1-mediated proteasomal degradation of the POI exclusively in those cells in G1/S phase during the treatment (Figure 1B).
Similarly, presence of OsTIR1-mEmerald-GEM (from now on: ROLECCS G2) protein would be restricted during the late S-G2-M phase, peaking during the G2, as APCCdh1-mediated ubiquitylation and degradation is rapidly triggered during M/G1 transition. Consequently, auxin treatment would cause degradation of the POI exclusively in cells going through the late S-G2-M phase of the cell cycle during the treatment (Figure 1C).
Each ROLECCS construct was abundantly expressed both transiently (at 72h from transfection) and stably (after AAVS1 knock-in) in HEK293 cells (Supplementary Figure 1B-D), both in the nucleus and in the cytoplasm of transfected cells. Live cell imaging was performed on ROLECCS AS, G1 and G2 knock-in HEK-293 to monitor the green fluorescence in real time. Figure 1D (top) and Supplementary Video 1 show that ROLECCS AS expression did not change during a full cell cycle. Conversely ROLECCS G1 was not visible in actively dividing cells (Figure 1D, middle and Supplementary Video 2), becoming detectable immediately upon completion of cell division. Finally, ROLECCS G2 was visible only in actively dividing cells, with the fluorescence intensity peaking at G2/M transition (Figure 1D, bottom and Supplementary Video 3).
To orthogonally validate ROLECCS G1 and ROLECCS G2 as cell cycle indicators, we sorted AAVS1-integrated ROLECCS HEK-293 based on their green fluorescence level and cellular complexity, as described in the methods section. GFPhigh-sorted ROLECCS G1 population mostly comprised cells in the G1/early S phase (94.6+1.6%), in comparison with GFPmed and GFPlow sorted populations (67.7+9.4% and 26.9+12.2% respectively) (Figure 1E-F). Conversely, GFPhigh ROLECCS G2 population showed a significant enrichment in late S/G2 phase cells (74.64+5%), compared to GFPmed and GFPlow sorted cells (28.8+7.2% and 3.5+1.5%, respectively) (Figure 1G-H). Conversely, unsorted ROLECCS G1 and ROLECCS G2 populations displayed cell cycle distribution typical of unsynchronized HEK-293 cells.
To assess that the enzymatic activity of OsTIR1-containing SCF complexes was not hampered by the mEmerald-Cdt1 and mEmerald-GEM tags of the ROLECCS G1 and G2, we transiently transfected AAVS1-integrated ROLECCS AS, ROLECCS G1, and ROLECCS G2 HEK-293 cells with a mAID-mCherry fluorescent reporter and measured its protein levels upon auxin treatment. mAID-mCherry levels were appreciably reduced upon auxin treatment when performed at 8h after reporter vector transfection, indicating that the biological activity of OsTIR1 was preserved (Supplementary Figure 1E). We hypothesized that an overexpressed target could be efficiently degraded only if the molar ratio between the POI and the ROLECCS was favorable to the latter, as in the very first hours (<8h) after transfection. Accordingly, other groups have generated All-in-One systems to achieve equimolar levels of OsTIR1 and its targets13.
As shown in Supplementary Figure 2A-B, we created 3 all-in-one lentiviral vectors (pLentiROLECCS AS, G1, and G2) in which the ROLECCS proteins were fused with mAID-mCherry using an autoproteolytic P2A sequence14.
Next, we treated HEK-293 cells stably expressing LentiROLECCS G1 or LentiROLECCS G2 with auxin for 1h. Cells were sorted based on their GFP fluorescence intensity and SSC (Supplementary Figure 3A-D). Red fluorescent intensity (from mAID-mCherry) was simultaneously quantified on GFPlow, GFPmed, and GFPhigh populations. Supplementary Figures 4A-B show that downregulation of the mCherry fluorescence was specifically achieved in GFPmed and GFPhigh populations upon auxin treatment.
Western blot analysis confirmed that GFPmed and GFPHigh sorted cell populations expressed the highest levels of ROLECCS G1 and ROLECCS G2 (Figure 1I-J and Supplementary Figure 4C-F). Notably, highest levels of ROLECCS G1 corresponded to highest expression of Cdt1 (a G1-specific marker, frequently identified as a doublet corresponding to Cdt1/phosphoCdt115) and to the lowest levels of Cyclin B1 (a late-S/G2 marker). Importantly, downregulation of the target mAID-mCherry was only observed in sorted GFPhigh ROLECCS G1 cells upon auxin treatment, and not in the untreated or GFPlow auxin-treated controls. Differently, ROLECCS G2 accumulation was observed in cell populations displaying highest levels of Cyclin B1 and lowest levels of Cdt1, but target downregulation was still only observed upon auxin treatment.
Taken together, our data indicate that the ROLECCS system allows temporally-restricted selective degradation of mAID-tagged targets based on the cell cycle phase.
Then, we decided to test whether the ROLECCS system could accomplish the cell cycle phase-specific downregulation of an endogenous target such as TP53, a well-known transcriptional factor playing a central role in the control of cell cycle progression, especially in response to DNA-damaging agents16,17.
First, we generated HCT116 cell lines where both wild-type TP53 alleles were modified by CRISPR/Cas9-mediated knock-in (HCT116 TP53-mAID-mCherry). For gene editing purposes, the stop codon of the endogenous TP53 gene was replaced by a mAID-mCherry fusion cassette (as described in Methods section) (Supplementary Figure 5A-D).
Western blot analysis showed that gene-edited TP53 had a marked molecular size increase (final predicted molecular weight ~87KDa, compared to WT TP53, 53KDa), due to the presence of the mAID and mCherry tags. Importantly, gene-edited TP53 was still upregulated by DNA damaging agents such as cisplatin treatment, and its nuclear and cytoplasmic localization followed the expected distribution pattern16,17 (Supplementary Figure 5E).
Next, we further edited HCT116 TP53-mAID-mCherry cells inserting the ROLECCS constructs in the AAVS1 safe harbor site, generating HCT116 TP53-mAID-mCherry ROLECCS cell lines (Figure 2A). As shown in Figure 2B, sustained expression of ROLECCS AS, G1, and G2 with the expected molecular weight was achieved in at least 2 independent clones. Since this analysis was performed on asynchronously growing HCT116 TP53-mAID-mCherry ROLECCS cells, the three ROLECCS constructs apparently displayed different expression levels. However, these differences are likely due to the fact that ROLECCS G1 and ROLECCS G2 are expressed only in phase-specific cell subpopulations, while ROLECCS AS is equally expressed throughout the cell cycle. Nonetheless, since we did not perform relative comparisons between ROLECCS AS and ROLECCS G1 or G2, these differences did not affect downstream analyses. We also noticed a mild reduction in the levels of edited TP53 in comparison with parental HCT116 cells, compatible with the partial leakiness observed for the AID system18,19. For this reason, for all the functional studies, cells were pre-treated with auxinole. a previously-reported inhibitor of OsTIR118, to neutralize the activity of the ROLECCS system in the absence of auxin.
Finally, we treated HCT116 TP53-mAID-mCherry ROLECCS G1 and G2 cells with auxin. At 1h after auxin treatment, cells were sorted (Supplementary Figure 6A-D) as described in the Methods section. As shown in Figure 2C-D, TP53 downregulation was noticeable in unsorted populations both in ROLECCS G1- and G2-expressing cells. However, upon sorting, we observed that TP53 downregulation upon auxin treatment was only achieved in GFPmed and GFPhigh sorted populations, in comparison with GFPlow cells for both ROLECCS constructs. Importantly, Cdt1 and Cyclin B1 levels confirmed that ROLECCS G1 GFPmed and GFPhigh represented a cell population enriched in G1/early S phase of the cell cycle. On the other hand, ROLECCS G2 GFPmed and GFPhigh cells were mostly representing cells in the late S/G2 phase. Similar results were obtained using two independent clones for each ROLECCS protein (Supplementary Figure 7A-D). These data indicate that the ROLECCS system can be used to achieve the phase-specific downregulation of an endogenous target, appropriately gene edited to include a mAID tag (Figure 2E).
Protein biological functions and cell cycle progression are intimately connected and reciprocally affected. Hence, the cell cycle status should be taken into account for the study of any biological phenomenon. Thanks to its phase specificity, rapidity, reversibility, and low overall perturbation of other biological processes, the ROLECCS technology represents a unique tool for the investigation of biological phenomena and their relationship with the cell cycle progression.