Integrated compact regulators of protein activity enable control of signaling pathways and genome-editing in vivo

Viral proteases and clinically safe inhibitors were employed to build integrated compact regulators of protein activity (iCROP) for post-translational regulation of functional proteins by tunable proteolytic activity. In the absence of inhibitor, the co-localized/fused protease cleaves a target peptide sequence introduced in an exposed loop of the protein of interest, irreversibly fragmenting the protein structure and destroying its functionality. We selected three proteases and demonstrated the versatility of the iCROP framework by validating it to regulate the functional activity of ten different proteins. iCROP switches can be delivered either as mRNA or DNA, and provide rapid actuation kinetics with large induction ratios, while remaining strongly suppressed in the off state without inhibitor. iCROPs for effectors of the NF-κB and NFAT signaling pathways were assembled and confirmed to enable precise activation/inhibition of downstream events in response to protease inhibitors. In lipopolysaccharide-treated mice, iCROP-sr-IκBα suppressed cytokine release (“cytokine storm”) by rescuing the activity of IκBα, which suppresses NF-κB signaling. We also constructed compact inducible CRISPR-(d)Cas9 variants and showed that iCROP-Cas9-mediated knockout of the PCSK9 gene in the liver lowered blood LDL-cholesterol levels in mice. iCROP-based protein switches will facilitate protein-level regulation in basic research and translational applications.


HRVp CS LEVLFQGP
Supplementary Table S3.Plasmids used and designed in this study.In vitro RNA production plasmid without mammalian promoter activity.PNF-κB-SEAP-pA PNF-κB-driven SEAP reporter protein expression vector.

pGM70
PhU6-sgRNAhINS PhU6-driven sgRNA complementary to a sequence in the human insulin promoter.

This work pNF214
PmPGK1-TetR-HIVp-VP64-pA Mammalian expression plasmid encoding PmPGK1-driven HIVp with its cleavage sites on C-end linked to C-terminal of DNA binding domain TetR fused to transcription activation domain VP64 with fusion linker containing NLS.
TetR was amplified from pTS1106 using oNF249 and oNF379.HIVp was amplified in a two-step process from Addgene plasmid #20253, first using oNF380 and oNF377 and second, using oNF380 and oNF358.PCR assembly reaction using oNF249 and oNF358 was used to fuse TetR and HIVp.The PCR fragment was digested with EcoRI/BamHI and ligated into pNF167 (EcoRI/BamHI).
sr-IκB was amplified from Addgene plasmid #15291, first with oNF383 and oNF384 and second with oNF385 and oNF390 to introduce CS, followed by PCR assembly reaction using oNF383 and oNF390.The PCR fragment was digested with EcoRI/XbaI and ligated into pNF167 (EcoRI/XbaI).
sr-IκB was amplified from Addgene plasmid #15291, first with oNF383 and oNF386 and second with oNF387 and oNF390 to introduce CS, followed by PCR assembly reaction using oNF383 and oNF390.The PCR fragment was digested with EcoRI/XbaI and ligated into pNF167 (EcoRI/XbaI).
This work sr-IκB was amplified from Addgene plasmid #15291, first with oNF383 and oNF388 and second with oNF389 and oNF390 to introduce CS, followed by PCR assembly reaction using oNF383 and oNF390.The PCR fragment was digested with EcoRI/XbaI and ligated into pNF167 (EcoRI/XbaI).pNF220 PmPGK1-sr-IκB-pA Mammalian expression plasmid encoding PmPGK1-driven sr-IκB.
HRVpQ182 was amplified from Twist gene fragment DNA_twist_HRVp using oNF353 and oNF403.TetR was amplified from pTS1106 using oNF260 and oNF261.PCR assembly reaction using oNF353 and oNF261 was used to fuse HRVpQ182 and TetR with 15gs linker.The PCR fragment was digested with EcoRI/BamHI and ligated into pNF167 (EcoRI/BamHI).
HRVpQ182 was amplified from Twist gene fragment DNA_twist_HRVp, first using oNF353 and oNF403, followed by second amplification using oNF353 and oNF358.The PCR fragment was digested with EcoRI/BamHI and ligated into pNF218 (EcoRI/BamHI).

This work pNF244
PmPGK1-HRVpQ182-TetR-CS-VP64-pA Mammalian expression plasmid encoding PmPGK1-driven HRVpQ182 linked to N-terminal of DNA binding domain TetR fused to transcription activation domain VP64 with fusion linker containing HRVp CS and NLS.
HRVp was amplified from Twist gene fragment DNA_twist_HRVp using oNF353 and oNF403.TetR was amplified from pTS1106 using oNF260 and oNF352.PCR assembly reaction using oNF353 and oNF352 was used to fuse HRVpQ182 and TetR-CS with 15gs linker.The PCR fragment was digested with EcoRI/BamHI and ligated into pNF167 (EcoRI/BamHI).
HRVpQ182 was amplified from pNF244 using oNF353 and oNF358 and digested with EcoRI/BamHI.HRVp CS was introduced into fLuc by amplifying BB3-fLuc first with oNF374 and oNF410 and second with oNF409 and oNF375 followed by PCR assembly reaction using oNF374 and oNF375.The PCR fragment was digested with BamHI/XbaI.Both fragments were ligated into pNF167 (EcoRI/XbaI).
MCP was amplified from pKK44 using oNF237 and oNF415.HRVpQ182-CS was amplified from pNF244 using oNF414 and oNF357.PCR assembly reaction using oNF237 and oNF357 was used to construct MCP-HRVpQ182-CS.The PCR fragment was digested with EcoRI/BamHI and ligated into pNF167 (EcoRI/BamHI).

This work pNF251
PmPGK1-HCVp-TetR-CS-VP64-pA Mammalian expression plasmid encoding PmPGK1-driven hepatitis C virus protease linked to N-terminal of DNA binding domain TetR fused to transcription activation domain VP64 with fusion linker containing HCVp cleavage site and NLS.
HCVp was amplified from Addgene plasmid #112628 using oNF468 and oNF469.TetR was amplified from pTS1106 using oNF260 and oNF471.PCR assembly reaction using oNF468 and oNF471 was used to fuse HCVp and TetR-CS with 15gs linker.
The PCR fragment was digested with EcoRI/BamHI and ligated into pNF167 (EcoRI/BamHI).
CS was inserted by amplifying pGM74 with oNF464/oNF465 and oNF466/oNF467, followed by PCR assembly reaction using oNF464 and oNF467.The PCR fragment was digested with BamHI/XbaI and ligated into pNF234 (BamHI/XbaI).
CS was inserted by amplifying pGM74 with oNF464/oNF531 and oNF530/oNF467, followed by PCR assembly reaction using oNF464 and oNF467.The PCR fragment was digested with BamHI/XbaI and ligated into pNF234 (BamHI/XbaI).
CS was inserted by amplifying pGM74 with oNF464/oNF533 and oNF532 and oNF467, followed by PCR assembly reaction using oNF464 and oNF467.The PCR fragment was digested with BamHI/XbaI and ligated into pNF234 (BamHI/XbaI).

This work pNF302
PmPGK1-TetR-HRVpQ182-CS-VP64-pA Mammalian expression plasmid encoding PmPGK1-driven HRVpQ182 with its cleavage sites on C-end linked to C-terminal of DNA binding domain TetR fused to transcription activation domain VP64 with fusion linker containing NLS.
TetR was amplified from pTS1106 using oNF249 and oNF379.HRVpQ182-CS was amplified from pNF244 using oNF355 and oNF357.PCR assembly reaction using oNF249 and oNF357 was used to fuse TetR and HRVpQ182-CS.The PCR fragment was This work digested with EcoRI/BamHI and ligated into pNF167 (EcoRI/BamHI).pNF303 PmPGK1-TetR-HRVp-VP64 Mammalian expression plasmid encoding PmPGK1-driven HRVp linked to C-terminal of DNA binding domain TetR fused to transcription activation domain VP64 with fusion linker containing NLS.
TetR was amplified from pTS1106 using oNF249 and oNF379.HRVp was amplified in a two-step process from pNF244, first using oNF355 and oNF354 and second using oNF355 and oNF358.PCR assembly reaction using oNF249 and oNF358 was used to fuse TetR and HRVp.The PCR fragment was digested with EcoRI/BamHI and ligated into pNF167 (EcoRI/BamHI).

This work pNF304
PmPGK1-TetR-CS-HCVp-CS-VP64 Mammalian expression plasmid encoding PmPGK1-driven HCVp with its cleavage sites on N-and C-end linked to C-terminal of DNA binding domain TetR fused to transcription activation domain VP64 with fusion linker containing NLS.
TetR was amplified from pTS1106 using oNF249 and oNF379.HCVp was amplified from Addgene plasmid #112628 using oNF477 and oNF478.PCR assembly reaction using oNF249 and oNF478 was used to fuse TetR and CS-HCVp-CS.The PCR fragment was digested with EcoRI/BamHI and ligated into pNF167 (EcoRI/BamHI).

This work pNF305
PmPGK1-TetR-HRVpQ182-VP64 Mammalian expression plasmid encoding PmPGK1-driven HRVpQ182 linked to C-terminal of DNA binding domain TetR fused to transcription activation domain VP64 with fusion linker containing NLS.
TetR was amplified from pTS1106 using oNF249 and oNF379.HRVpQ182 was amplified from pNF244 using oNF355 and oNF358.PCR assembly reaction using oNF249 and oNF358 was used to fuse TetR and HRVpQ182.The PCR fragment was digested with EcoRI/BamHI and ligated into pNF167 (EcoRI/BamHI).
MCP was amplified from pKK44 using oNF237 and oNF415.HRVp was amplified from pNF303 using oNF414 and oNF358.PCR assembly reaction using oNF237 and oNF358 was used to construct MCP-HRVp.The PCR fragment was digested with EcoRI/BamHI and ligated into pNF167 (EcoRI/BamHI).
N-terminal part of fLuc was amplified from BB3-fLuc using oNF374 and oNF534.HRVp was amplified from pNF303 using oNF408 and oNF354.C-terminal part of fLuc was amplified from BB3-fLuc using oNF545 and oNF375.HRVp and C-fLuc were fused by PCR assembly using oNF408 and oNF375, followed by another PCR assembly to add N-fLuc using oNF374 and oNF375.

This work pNF322
PhU6-sgRNAEMX1 PhU6-driven sgRNA complementary to a sequence in the human EMX1 gene.
CS was inserted by amplifying pNF286 with oNF464/oNF533 and oNF532/oNF467, followed by PCR assembly reaction using oNF464 and oNF467.The PCR fragment was digested with BamHI/XbaI and ligated into pNF234 (BamHI/XbaI).
CS was inserted by amplifying pNF334 with oNF464/oNF531 and oNF530 and oNF467, followed by PCR assembly reaction using oNF464 and oNF467.The PCR fragment was digested with BamHI/XbaI and ligated into pNF218 (BamHI/XbaI).
CS was inserted by amplifying pNF334 with oNF464/oNF465 and oNF466 and oNF467, followed by PCR assembly reaction using oNF464 and oNF467.The PCR fragment was digested with BamHI/XbaI and ligated into pNF218 (BamHI/XbaI).

This work pNF359
PhU6-sgRNAVEGFA-1 P hU6 -driven sgRNA complementary to a sequence in the exon 4 of human VEGFA gene.

This work pNF360
PhU6-sgRNAVEGFA-2 PhU6-driven sgRNA complementary to a sequence in the exon 5 of human VEGFA gene.

This work pNF361
PhU6-sgRNAVEGFA-3 PhU6-driven sgRNA complementary to a sequence in the exon 8 of human VEGFA gene.

This work pNF362
PhU6-sgRNATNFRSF1A-1 This work PhU6-driven sgRNA complementary to a sequence in the exon 8 of human TNFRSF1A gene.

This work pNF364
PhU6-sgRNATNFRSF1A-3 PhU6-driven sgRNA complementary to a sequence in the exon 8 of human TNFRSF1A gene.

This work pNF365
PhU6-sgRNAACE2-1 PhU6-driven sgRNA complementary to a sequence in the exon 18 of human ACE2 gene.

This work pNF366
PhU6-sgRNAACE2-2 PhU6-driven sgRNA complementary to a sequence in the exon 13 of human ACE2 gene.

This work pNF367
PhU6-sgRNAACE2-3 PhU6-driven sgRNA complementary to a sequence in the exon 14 of human ACE2 gene.

This work pNF368
PhU6-sgRNAPCSK9 PhU6-driven sgRNA complementary to a sequence in the exon 1 of murine PCSK9 gene.
This work pNF372 P mPGK1 -RelA-pA Mammalian expression plasmid encoding PmPGK1-driven full length RelA.
RelA was first amplified from pRSV-p65 using oNF660 and oNF662 and second with oNF661 and oNF663 to remove internally occurring EcoRI site, following by PCR assembly using oNF660 and oNF663.The PCR fragment was digested with EcoRI/XbaI and ligated into pNF167 (EcoRI/XbaI) This work pNF377 P mPGK1 -HCVp-fLuc CS-K491 -pA Mammalian expression plasmid encoding PmPGK1-driven HCVp linked to N-terminal of fLuc containing HCVp CS.
HIVp was amplified from pNF212 using oNF376 and oNF358 and digested with EcoRI/BamHI.HIVp CS was introduced into fLuc by amplifying BB3-fLuc first with oNF374/oNF659 and second with oNF658/oNF375 followed by PCR assembly reaction using oNF374 and oNF375.PCR fragment was digested with BamHI/XbaI.Both fragments were ligated into pNF167 (EcoRI/XbaI).
RelAN-term was amplified from pNF372 using oNF660 and oNF664 and HRVpQ182-CS was amplified from pNF288 using oNF408 and oNF357, followed by PCR assembly reaction with oNF660 and oNF357.The PCR fragment was digested with EcoRI/BamHI.RelAC-term was amplified from pNF372 using oNF665 and oNF663 and digested with BamHI/XbaI.Both fragments were ligated into pNF167 (EcoRI/XbaI).
The PCR product was digested with EcoRI/XbaI and ligated into pNF167 (EcoRI/XbaI).
RelAN-term was amplified from pNF372 using oNF660 and oNF664 and HRVp was amplified from pNF311 using oNF408 and oNF358, followed by PCR assembly reaction with oNF660 and oNF358.The PCR fragment was digested with EcoRI/BamHI.RelAC-term was amplified from pNF372 using oNF665 and oNF663 and digested with BamHI/XbaI.Both fragments were ligated into pNF167 (EcoRI/XbaI).
HRVpQ182 was excised from pNF234 with EcoRI/BamHI.To introduce CS, MyD88 was first amplified from MYD88 using oNF689 and oNF673 and second with oNF674 and oNF670, followed by PCR assembly reaction using oNF689 and oNF670 and digestion with BamHI/XbaI.Both fragments were ligated into pNF167 (EcoRI/XbaI).
HRVpQ182 was excised from pNF234 with EcoRI/BamHI.To introduce CS, MyD88 was first amplified from MYD88 using oNF689 and oNF675 and second with oNF676 and oNF670, followed by PCR assembly reaction using oNF689 and oNF670 and digestion with BamHI/XbaI.Both fragments were ligated into pNF167 (EcoRI/XbaI).
To introduce CS, MyD88 was first amplified from MYD88 using oNF689 and oNF671 and second with oNF672 and oNF670, followed by PCR assembly reaction using oNF689 and oNF670, and digestion with BamHI/XbaI.The fragment was ligated into pNF220 (BamHI/XbaI).
To introduce CS, MyD88 was first amplified from MYD88 using oNF689 and oNF673 and second with oNF674 and oNF670, followed by PCR assembly reaction using oNF689 and oNF670 and digestion with BamHI/XbaI.The fragment was ligated into pNF220 (BamHI/XbaI).
To introduce CS, MyD88 was first amplified from MYD88 using oNF689 and oNF675 and second with oNF676 and oNF670, followed by PCR assembly reaction using oNF689 and oNF670 and digestion with BamHI/XbaI.The fragment was ligated into pNF220 (BamHI/XbaI).

Figure S3 :
Figure S3: Performance of different iCROP systems applied to the multi-domain protein TetR-VP64.a) Scheme of TetR-VP64 modified to contain a protease cleavage site in the linker between TetR and VP64 and the corresponding protease (HIVp, HCVp or HRVp) fused to its N-terminus.b-d) SEAP secretion from HEK293T cells expressing each TetR-VP64 protease system, and treated for 24 h with different concentrations of the corresponding inhibitors.e) SEAP secretion by HEK293T cells with genomic integration of the expression cassettes OTetR-PhCMVmin-SEAP and PmPGK-HRVpQ182-TetR-CS-VP64 (pNF417).The HEK-iCROP cells were

Figure S4 :
Figure S4: Effect of modifications on TetR-VP64 and fLuc activities.a) SEAP secretion by cells co-transfected with OTetR-PhCMVmin-SEAP (pNF293) and with constitutive expression of either unmodified TetR-VP64 or TetR-VP64 modified as indicated (either with a cleavage site or the HRVQ182 protease between the TetR and VP64 domains or the HRVQ182 protease Nterminally fused to TetR).SEAP expression levels were quantified 48 h post-transfection.b) Luminescence of cells transfected with unmodified fLuc or fLuc bearing the HRV cleavage site downstream of K491, either without or with the HRV Q182 protease N-terminally fused.Following transfection, the luminescence intensity was quantified 48 h thereafter.Data are shown as mean ± s.d., with individual data points (n = 3 biological replicates).Statistical significance was calculated by means of Welch's two-tailed t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant.

Figure S5 :
Figure S5: Assessment of various iCROP-fLuc topologies, kinetics and reversibility.a) fLuc 3D structure (PDB: 1BA3).The arrow points to residue K491, where the CS was placed.b) Bioluminescence intensity of HEK293T cells transfected with fLuc harboring both HRVp and CS downstream of K491, and incubated with the indicated rupintrivir concentrations for 24 h.c) Bioluminescence intensity of HEK293T cells co-transfected with fLuc bearing the CS downstream of K491 and a separate plasmid encoding HRVp.The cells were then incubated with the indicated rupintrivir concentrations for 24 h.d) Comparison of the three strategies for control of fLuc activity based on rupintrivir inhibition of HRVp.The fold change was calculated by dividing the fLuc activity at each rupintrivir concentration by the fLuc activity of untreated cells.e) Kinetics of iCROP-fLuc.Time-course analysis of fLuc luminescence in HEK293T cells transfected with iCROP-fLuc (PmPGK-HRVp Q182 -fLucCS-K491-pA) and incubated for 48 h either in the absence or presence of rupintrivir (1 µM).f) iCROP-fLuc reversibility.HEK293T cells expressing iCROP-fLuc were alternated between rupintrivir-containing (100 nM) and rupintrivir-free media.Each rupintrivir induction lasted 4 h, followed by 12 h in rupintrivir-free medium.fLuc intensity was measured at the indicated time points.Data in panels b-f are shown as mean ± s.d., with individual data points in panels b-d (n = 3 biological replicates).Statistical significance was calculated by means of Welch's two-tailed t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant.

Figure S7 :
Figure S7: Design of inducible NF-κB regulators.a) Predicted 3D structure of MyD88 (AlphaFold: AF-Q99836-F1).The arrow points to residue G83, selected as the best location to place the CS.b) NF-κB activation by overexpression of wild-type (WT) and CS-modified MyD88 variants.SEAP levels were analyzed 36 h after co-transfection of HEK293T cells with constitutive expression of each MyD88 variant and SEAP under an NF-κB-responsive promoter (PNF-κB-SEAP-pA).Cells transfected with the reporter only were included as a negative control.

Figure S8 :
Figure S8: Design and optimization of inducible NFAT regulators.a) Schematic illustration of representative NFAT structure.The arrow points to the region of the DNA binding domain where the HRVp and CS were placed.b) Activation of SEAP expression from an NFAT promoter (pMX57, P3xNFAT-SEAP-pA) in cells co-transfected with different NFAT1 variants, namely wt NFAT1, truncated NFAT1S172-T925, and iCROP-NFAT1.Cells transfected with

Figure S15 :
Figure S15: Effect of rupintrivir on PCSK9 and LDL-cholesterol in vivo.Blood levels of a) PCSK9 and b) LDL-cholesterol in WT mice that were either non-treated or treated with rupintrivir.Data are shown as mean ± s.d., with individual data points (n = 5 mice per group).Statistical significance was calculated by means of Welch's two-tailed t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant.