Achieving tight control of a photoactivatable Cre recombinase gene switch: new design strategies and functional characterization in mammalian cells and rodent

Abstract

A common mechanism for inducibly controlling protein function relies on reconstitution of split protein fragments using chemical or light-induced dimerization domains. A protein is split into fragments that are inactive on their own, but can be reconstituted after dimerization. As many split proteins retain affinity for their complementary half, maintaining low activity in the absence of an inducer remains a challenge. Here, we systematically explore methods to achieve tight regulation of inducible proteins that are effective despite variation in protein expression level. We characterize a previously developed split Cre recombinase (PA-Cre2.0) that is reconstituted upon light-induced CRY2-CIB1 dimerization, in cultured cells and in vivo in rodent brain. In culture, PA-Cre2.0 shows low background and high induced activity over a wide range of expression levels, while in vivo the system also shows low background and sensitive response to brief light inputs. The consistent activity stems from fragment compartmentalization that shifts localization toward the cytosol. Extending this work, we exploit nuclear compartmentalization to generate light-and-chemical regulated versions of Cre recombinase. This work demonstrates in vivo functionality of PA-Cre2.0, describes new approaches to achieve tight inducible control of Cre DNA recombinase, and provides general guidelines for further engineering and application of split protein fragments.

Figure 1.

Alteration of expression level of CRY2-CreN and CIB1-CreC fragments causes changes in dark background, with minimal effects on light-induced activity. (A) Schematic showing combined constructs used in (B) and (C). (B) Quantification of Cre recombinase activity in HEK293T cells. Cells expressing CRY2(L348F)-CreN in front of an IRES element (KM60) show higher dark background compared with KM10. Cells were transfected with indicated plasmids and a loxP-STOP-loxP-EGFP reporter, then kept in dark and harvested at 48 h. Light-treated samples were exposed to a 4 s pulse of light at 24 h. Graph shows average and error (s.d., n = 4–6 independent experiments). (C) Immunoblot comparing expression of Cre fragments in KM10 versus KM60 (anti-Cre antibody, top panel; anti-tubulin, bottom panel). (D) Immunoblots of dual plasmid system. HEK293 cells were transfected with indicated constructs and immunoblotted with an anti-Cre antibody (top). Bottom panel shows anti-tubulin loading control. Control lane consisted of non-transfected cells. (E) Quantification of Cre recombinase activity in HEK293T cells transfected with indicated constructs and light-treated as in (B). Protein expression level as determined in (D) is indicated below the graph, with ‘+’ (low expression) or ‘+++’ (high expression). Graph shows average and error (s.d., n = 3–5 independent experiments). (ns, not significant; *, P < .05, student's t-test). (F) Comparison of expression level and activity of HEK293T cells expressing a combined PA-Cre2.0 construct (KM10), or an identical version (ΔpA) with a mutant polyadenylation signal. Expression of the Cre fragments is greatly reduced in the ΔpA version. Dark background activity is significantly reduced (*, P < .05), but no significant difference in light-stimulated activity is observed. Graph shows average and error (s.d., n = 3–6 independent experiments). (G) Quantification of Cre recombinase activity with different amounts of wt or L348F CRY2-CreN. All samples contained 1 μg EGFP Cre reporter and 1 μg p645 (CIB1-CreC) and were quantified 48 h post-transfection. Light samples received a 4 h light exposure at 24 h (2 s pulse every 1 min, 461 nm). Samples labeled ‘high’ were transfected with 1 μg of L348F or wt CRY2-CreN; ‘low’ samples contained 50 ng. Graph shows average and range of two independent experiments.

Figure 2.

CRY2 L348F mutant shows reduced nuclear localization. (A) Reduced dark activity of CRY2(L348F)-CreN is not due to reduced overall expression. Shown is an immunoblot of cells (anti-myc antibody) expressing myc-tagged versions of wild-type or L348F CRY2-CreN. Loading control, Ponceau S total protein reference band. (B) Localization of mCherry-fused versions of CRY2 (wild-type) or CRY2 L348F. The L348F mutation reduces the percent of expressed protein localized to the nucleus. Graph at right shows quantification of the ratio of nuclear:cytosolic protein (average and error, s.e.m, n = 7 cells; *, P < .05, student's t-test). Scale bars, 10 μm. (C) mCherry-fused versions of L348F CRY2-CreN show even further reduced nuclear localization, compared to wt. Mutation of a putative ‘NES’ in CreN (CreN ΔNES) or addition of an extra NLS (far right, CRY2-L348F-NLS) restores nuclear localization. Graph at right shows quantification of nuclear:cytosolic protein (average and error, s.e.m., n = 7 cells (10 cells for ΔNES); *, P < .05, student's t-test). Scale bars, 10 μm. (D) Quantification of Cre recombinase activity. Addition of an extra NLS (to generate CRY2(L348F)-NLS-CreN) results in enhanced dark activity, with minimal effects on light activity. Graph represents average and range from two independent experiments. (E) Localization of switched CreN and CreC versions. While CRY2(L348F) combined with CreN shows reduced nuclear localization, switched CRY2(L348F)-CreC shows strong nuclear localization. The CIB1 construct shows strong nuclear localization with either CreC or CreN. Scale bars, 10 μm. (F) Quantification of Cre recombinase activity in cells expressing CRY2(L348F)-CreN/CIB1-CreC (‘original’) or CRY2(L348F)-CreC/CIB1-CreN (‘switched’) versions of PA-Cre. Both mCherry-fused and non-fused versions of CIB1-CreN were assayed and results combined. Data represents average and error (s.d., n = 4–6 independent experiments; P < .05, student's t-test). (G) Quantification of nuclear fluorescence relative to total cell fluorescence. HEK293T cells were transfected with increasing amounts of wt or L348F mCh-CRY2-CreN. The concentration of mCherry fluorescent signal in the nucleus, relative to total protein abundance, was quantified. Each dot represents an individual cell, arbitrary units. Red triangles, wt; blue circles, L348F. At all concentrations, wt expressing cells show higher nuclear signal compared with L348F. (H) Model of CRY2(L348F)-CreN nuclear trafficking. The CreN fragment contains a sequence recognized as a NES, while the L348F mutation appears to impair CRY2 nuclear localization. Together this results in a protein with reduced nuclear occupancy.

Figure 3.

Functional comparison of CRY2/CIB1 PA-Cre2.0 with Mag PA-Cre. (A) Comparison of PA-Cre2.0 and Mag PA-Cre activity at different expression levels. HEK293T cells were transfected with either 1 μg of each indicated Cre system (KM69 or KM91) and 1 μg DIO-EGFP reporter (1:1 ratio), or with 50 ng each Cre system and 450 ng reporter (1:9 ratio). Samples were treated with dark or 4 h light at 24 h, before quantifying at 48 h. Graph indicates average and range of two independent experiments. (B) Comparison of PA-Cre2.0 and Mag PA-Cre responses to a 4 s light pulse. HEK293T cells were transfected with 1 μg of PA-Cre2.0 (KM69) and 1 μg DIO-EGFP or loxP-STOP-loxP-EGFP reporter (1:1), or 50 ng of Mag PA-Cre (KM91) and 450 ng of each reporter (1:9). Samples were treated with dark or light as in (A) and quantified at 48 h. Graph indicates average and range of two independent experiments.

Figure 4.

In vivo validation of PA-Cre2.0. (A) AAVs (hSyn-EYFP, CaMKII-mycCRY2(L348F)-CreN and CaMKII-CIB1-CreC) were mixed at a 1:1:1 ratio and injected into the dentate gyrus of Ai14 reporter mice. (B) Mice were implanted with an optical fiber (dashed outline in Figure 2C) over hippocampus. (C) Representative images from control animals (no light pulse, top row) or animals exposed to a 1 s pulse of blue light (473 nm) (‘Light’, bottom row). EYFP (green) marks virally infected neurons, with DAPI nuclear staining highlighted in blue. Minimal tdTomato expression (red) was observed in control animals (n = 3) that did not receive light, while light-treated animals (n = 3) showed Cre-dependent tdTomato expression in CaMKII positive cells in brain. Scale bar, 400 μm.

Figure 5.

Light and chemical regulation of Cre recombinase activity. (A) Schematic of TMP-regulated PA-Cre, in which destabilized Escherichia coli DHFR is fused to the CRY2(L348F)-CreN fragment. (B) HEK293T cells were transfected with the constructs indicated in (A) (CW79, 1 μg; p645, CIB1-CreC, 1 μg), along with a loxP-STOP-loxP-EGFP reporter (1 μg). Twenty-four hours after transfection, TMP (10 μM) was added to indicated samples. After 30–120 min, samples were maintained in dark or exposed to light (461 nm pulse, 2 s every 2 min for 30 min) then quantified at 48 h. Graph represents average and error (s.d., n = 3 independent experiments). (C) Schematic of estradiol-and-light-regulated Cre recombinase, containing an ERα-LBD fused to CRY2(L348F)-CreN. (D) HEK293T cells were transfected with the constructs indicated in (C) (CW57, 1 μg; p645, CIB1-CreC, 1 μg), and a loxP-STOP-loxP-EGFP reporter (1 μg). Twenty-four hours after transfection, 17-β-estradiol (400 nM) was added to indicated samples, then samples were kept in dark or exposed to 4 h light (461 nm pulse, 2 s every 3 min), then quantified at 48 h. Control samples labeled ‘No LBD’ contained p688, p645 and the reporter. Graph represents average and range of two independent experiments.