A robust dual gene ON–OFF toggle directed by two independent promoter–degron pairs

ABSTRACT Switching genes on and off on cue is a cornerstone for understanding gene functions. One contemporary approach for loss-of-function studies of essential genes involves CRISPR-mediated knockout of the endogenous locus in conjunction with the expression of a rescue construct, which can subsequently be turned off to produce a gene inactivation effect in mammalian cell lines. A broadening of this approach would involve simultaneously switching on a second construct to interrogate the functions of a gene in the pathway. In this study, we developed a pair of switches that were independently controlled by both inducible promoters and degrons, enabling the toggling between two constructs with comparable kinetics and tightness. The gene-OFF switch was based on TRE transcriptional control coupled with auxin-induced degron-mediated proteolysis. A second independently controlled gene-ON switch was based on a modified ecdysone promoter and mutated FKBP12-derived destabilization domain degron, allowing acute and tuneable gene activation. This platform facilitates efficient generation of knockout cell lines containing a two-gene switch that is regulated tightly and can be flipped within a fraction of the time of a cell cycle.

(A) Aberrant expression of truncated products from the mIAA7 degron. HeLa cells stably expressing either mAID CDK2 or mIAA7 CDK2 together with TIR1 or AFB2 (both myc-tagged), respectively, were generated. The endogenous CDK2 was at the same time disrupted with CRISPR-Cas9. The cells were cultured in either the presence or absence of Dox and IAA (DI) for 24 h to silence the degron-tagged CDK2. Lysates were prepared and analyzed with immunoblotting. Lysates from HeLa were also loaded to indicate the expression of endogenous CDK2. Note the presence of a truncated product from mIAA7 CDK2 (∆), which is slightly bigger than endogenous CDK2. Equal loading of lysates was confirmed by immunoblotting for actin. (B) mIAA7 possesses an internal methionine that is absent in mAID. Alignment of the amino acid sequences between mAID and mIAA7 is shown. Identical amino acids are highlighted in grey. Met 52 of mIAA7 and Val 36 in mAID are highlighted in yellow.
(C) Mutation of Met 52 largely abolishes the expression of truncated products from mIAA7. Point mutation M52V was introduced into mIAA7 to generate mIAA7(MV).
Cell lines expressing mAID-, mIAA7-, or mIAA7(MV)-tagged CDK2 in combination with TIR1 or AFB2 were either untreated or incubated with DI for 24 h before analyzed with immunoblotting. The lower bands in the actin blot were signals from the previous CDK2 blot. Cell lines expressing mIAA7-or mIAA7(MV)-tagged CDK2 in combination with AFB2 were generated. The cells were incubated with IAA and harvested at the indicated time points immunoblotting analysis. Equal loading of lysates was confirmed by immunoblotting for actin. (B) Degrons (mAID and mIAA7) and F-box proteins (TIR1 and AFB2) are interchangeable. HeLa cells expressing mAID CDK2 and AFB2 or mIAA7(MV) CDK2 with TIR1 were generated (endogenous CDK2 was also disrupted with CRISPR-Cas9).
The cells were treated with Dox and IAA individually or together for 24 h before analyzed with immunoblotting.
(C) Trace amounts of truncated products is still present in M52V-mutated mIAA7.
Cell lines expressing mAID-or mIAA7(MV)-tagged CDK2 in combination with TIR1 or AFB2, respectively, were generated. The cells were incubated with DI and harvested at the indicated time points for immunoblotting analysis. (D) Relatively low background degradation for mAID-tagged proteins in the presence of AFB2. Cell lines expressing the indicated combination of mAID CDK2, mIAA7(MV) CDK2, TIR1, and AFB2 were treated with cycloheximide (CHX) to abolish de novo protein synthesis. The cells were harvested at different time points and analyzed with immunoblotting.
(E) mIAA7 has a higher background degradation rate than mAID. Cell lines expressing different configurations of mAID-or mIAA7(MV)-tagged CDK2 and TIR1 or AFB2 were generated. Protein expression was analyzed using immunoblotting (upper panel). The relative expression of recombinant CDK2 mRNA was quantified using RT-PCR (lower panel; same primers were used to detect both mAID-and mIAA7(MV)-tagged CDK2 mRNAs). Mean ± SEM from three independent experiments.  The pUHD-SB-mAID series were as described in , containing TRE-driven mAIDfusion cDNA (X) placed in between the ITRs of SB transposon. The pSBbi-AFB2/Pur vector contains AFB2 driven by a constitutive EF1α promoter (also for SB transposon). The pSBbi-AFB2-tTA/Neo vector contains AFB2 driven by a constitutive EF1α promoter and tTA driven by a RPBSA promoter.
pAFB2-H2B-Clover/Zeo expresses both AFB2 and histone H2B-Clover placed in between the 5' homology arm (HA-L) and 3' homology arm (HA-R) of AAVS1. Antibiotic resistance genes for blasticidin (Bla R ), hygromycin (Hyg R ), puromycin (Pur R ), zeocin (Zeo R ), or neomycin (Neo R ) are driven by a RPBSA promoter. T2A: T2A ribosomal skipping site. (C) Sustained activation of DD-tagged proteins. CDK2 DD -expressing cells in a CDK2-KO background were either untreated or incubated with PS. Lysates were prepared on different days and analyzed with immunoblotting. Note that cells were plated initially at low density; and PS was not replenished during the experiment.  The pDBEcR series of vectors contain pDBEcR (myc-tagged) driven by a constitutive EF1α promoter and placed in between the ITRs of SB transposon. The pDBEcR-pIND(SP1) series further include the gene of interest (Y) under the control of a promoter containing E/GRE (ecdysone/glucocorticoid response element x6), SP1 (Specificity Protein 1 enhancer x3), and P∆HSP (minimal heat shock protein promoter). Some versions of the vectors contain DD degron (mutated FKBP12-derived destabilization domain degron) fused to the C-terminus of "Y". All available vectors contain EGFP as "Y", which can be excised to replace with a gene of interest. Blasticidin (Bla R ) resistance gene is driven either by a RPBSA promoter or T2A ribosomal skipping site. Some versions of the vectors contain WPRE at the 3'-UTR of "Y". Sequence information of pDBEcR-pIND(SP1)/Bla, pDBEcR-pIND(SP1)-W/Bla, pDBEcR-pIND(SP1)-DD/Bla, and pDBEcR-pIND(SP1)-DD/Bla are shown below.

pDBEcR-pIND(SP1)/Bla
Region containing FLAG (grey) and EGFP (green). Unique restriction enzyme sites are indicated. Cell Sci.: doi:10.1242/jcs.260754: Supplementary information Graphic map, cloning sites, locations of different elements, and complete DNA sequence. The vector expresses FLAG-tagged EGFP under the control of a modified ecdysone promoter. DBEcR (myc-tagged at C-terminus) and blasticidin resistance gene are expressed from a constitutive EF1α promoter. FLAG-EGFP can be excised with NheI and NotI/EcoRI and replaced with other cDNAs.

pDBEcR-pIND(SP1)-DD-W/Bla
(E) Model of the relationship between CDK1 and CDK2. The relationship between the expression of CDKs in KO cell lines according to   [Cell Reports 37: 109808]. In normal HeLa cells, CDK1 and CDK2 are responsible in driving G2/M and G1/S, respectively. In the absence of CDK2, endogenous CDK1 is able to take over the role CDK2. However, CDK2 is insufficient to replace CDK1's G2/M functions unless overexpressed (OE).
(F) Exchanging WT with kinase-dead mutant of CDK2. Two CDK2 KO cell lines expressing mAID CDK2 and either WT or K33R CDK2 DD were generated. The cells were treated with DI (to turn off mAID CDK2) and PS (to turn on CDK2 DD ) for 24 h.
Lysates were prepared and subjected to immunoprecipitation with cyclin A antibodies. Both total cell lysates and immunoprecipitates were analyzed with immunoblotting. The parental HeLa cells were also loaded as controls (containing endogenous CDK2). CDK1 and CDK2 were detected with both specific antibodies and a PSTAIRE monoclonal antibody, which recognizes an epitope present in both proteins. Endogenous CDK1 and CDK2 are similar in size. As lanes 2-7 do not contain endogenous CDK2, the PSTAIRE signals were from CDK1. Ligation of NheI-NcoI-cut PCR product (primers 1-2; template: pSH-EFIRES-P-Seipin-miniIAA7-3XFlag (a gift from Elina Ikonen; Addgene #129722)) into NheI-NcoI-cut pUHD-SB-AID/Hyg .