Machine-Guided Dual-Objective Protein Engineering for Deimmunization and Therapeutic Functions
Wolfsberg E, Paul J-P, Tycko J, Chen B, Bassik MC, Bintu L, Alizadeh AA, Gao XJ
bioRxiv 2025.02.24.639918
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Article
Plasmids from Article
| ID | Plasmid | Purpose |
|---|---|---|
| 236119 | PGK-mCherry | Fluorescent transfection marker encoding mCherry under PGK promoter |
| 236120 | CMV-TO-g0 (FLP-IN) | Plasmid encoding g0 guide RNA under control of CMV promoter with two TetR binding sites, used to equalize plasmid masses for transfection |
| 236121 | EK0007-PB-4XnrUAS-H2B-BFP | Fuorescent reporter encoding H2B-tagBFP fusion under control of E1b minimal promoter with four Gal4-binding sites upstream |
| 236122 | EW269 CMV-TO-Gal4-NZF[GS Linkers] (FLP-IN) | Plasmid encoding Gal4-NZF transcription factor with GS linkers between the component domains of NZF, under control of CMV promoter with two TetR binding sites |
| 236123 | EW152 CMV-TO-Gal4-NZF[Mutated Linkers] (FLP-IN) | Plasmid encoding Gal4-NZF transcription factor with deimmunized linkers between the component domains of NZF, under control of CMV promoter with two TetR binding sites |
| 236124 | EW265 SFFV-mCherry-SGc(stem-1(3xPUF9R-tar))cSG-EGFP (FLP-IN) | Plasmid encoding fluorescent reporter for PUF-9R binding which expresses mCherry constitutively and EGFP upon stop codon deamination due to ADAR-PUF9R binding |
| 236125 | EW446 CMV-TO-ADAR2dd(E488Q, T501A) (FLP-IN) | Plasmid encoding free ADAR2dd under control of CMV promoter with two TetR binding sites |
| 236126 | EW263 CMV-TO-ADAR2dd(E488Q, T501A)-PUF-9R(FLP-IN) | Plasmid encoding ADAR2dd attached to PUF-9R under control of CMV promoter with two TetR binding sites |
| 236127 | EW267 CMV-TO-ADAR2dd(E488Q, T501A)-PUF-9R-MARIAcomp(V241D, H243M) (FLP-IN) | Plasmid encoding ADAR2dd attached to PUF-9R with deimmunizing mutations under control of CMV promoter with two TetR binding sites |
| 236128 | EW482 CMV-TO-ADAR2dd(E488Q, T501A)-PUF-9R-MARIAcomp(V241D, H243M) [D241C, M243Q] (FLP-IN) | Plasmid encoding ADAR2dd attached to PUF-9R with deimmunizing and function-restoring mutations under control of CMV promoter with two TetR binding sites |
| 236129 | EW408 (ZNF35tar-compact)x8-H2B-GFP | Fuorescent reporter encoding H2B-GFP fusion under control of E1b minimal promoter with eight ZNF35 binding sites upstream |
| 236130 | EW215 PGK-ZNF35(5-8)-deImmunLink-NZF(FLP-IN) | Plasmid encoding the ZNF35-derived portion of the ZNF35/ZNF250 zinc finger array attached by a deimmunized linker to the NZF transcriptional activation domain, under control of PGK promoter |
| 236131 | EW216 PGK-ZNF250(1-4)-deImmunLink-NZF(FLP-IN) | Plasmid encoding the ZNF250-derived portion of the ZNF35/ZNF250 zinc finger array attached by a deimmunized linker to the NZF transcriptional activation domain, under control of PGK promoter |
| 236132 | EW196 PGK-ZNF35(5-8)-ZNF250(1-4)-deImmunLink-NZF(FLP-IN) | Plasmid encoding the ZNF35/ZNF250 zinc finger array attached by a deimmunized linker to the NZF transcriptional activation domain, under control of PGK promoter |
| 236133 | EW409 ZNF250tarv3 x8-H2B-GFP | Fuorescent reporter encoding H2B-GFP fusion under control of E1b minimal promoter with eight ZNF250 binding sites upstream |
| 236134 | EW378 (ZNF35/ZNF250tarv3-6bp spacers)x4-H2B-GFP | Fuorescent reporter encoding H2B-GFP fusion under control of E1b minimal promoter with four ZNF35/ZNF250 binding sites upstream |
| 236135 | EW209 PGK-ZNF35(5-8)-ZNF35(5-8)-deImmunLink-NZF(FLP-IN) | Plasmid encoding the ZNF35/ZNF35 zinc finger array attached by a deimmunized linker to the NZF transcriptional activation domain, under control of PGK promoter |
| 236136 | EW415 PGK-ZNF35(5-8)-IKZF1(2-3)-realDeimmunLink-NZF (FLP-IN) | Plasmid encoding the ZNF35/ZNFIKZF1 zinc finger array attached by a deimmunized linker to the NZF transcriptional activation domain, under control of PGK promoter |
| 236137 | EW407 (ZNF35/ZNF35-6bp spacers)x4-H2B-GFP | Fuorescent reporter encoding H2B-GFP fusion under control of E1b minimal promoter with four ZNF35/ZNF250 binding sites upstream |
| 236138 | EW387 (ZNF35/IKZF1tar-6bp spacers)x4-H2B-GFP | Fuorescent reporter encoding H2B-GFP fusion under control of E1b minimal promoter with four ZNF35/ZNFIKZF1 binding sites upstream |
| 236139 | EW420 4x(ZNF35/ZNF250tarv3)-SFFV-GFP | Fluorescent reporter encoding GFP under control of SFFV promoter with four ZNF35/ZNF250 binding sites upstream |
| 236140 | EW416 CMV-TO-ZNF35(5-8)-ZNF250(1-4)-deImmLink-ZIK1KRAB | Plasmid encoding the ZNF35/ZNF250 zinc finger array attached by a deimmunized linker to the ZIK1 KRAB domain, under control of CMV promoter with two TetR binding sites |
| 236141 | EW428 CMV-TO-ZNF35(5-8)-ZNF35(5-8)-deImmLink-ZIK1KRAB | Plasmid encoding the ZNF35/ZNF35 zinc finger array attached by a deimmunized linker to the ZIK1 KRAB domain, under control of CMV promoter with two TetR binding sites |
| 236142 | EW425 CMV-TO-ZNF35(5-8)-IKZF1(2-3)-realDeImmLink-ZIK1KRAB | Plasmid encoding the ZNF35/IKZF1 zinc finger array attached by a deimmunized linker to the ZIK1 KRAB domain, under control of CMV promoter with two TetR binding sites |
| 236143 | EW434 4x(ZNF35/ZNF35tar)-SFFV-GFP | Fluorescent reporter encoding GFP under control of SFFV promoter with four ZNF35/ZNF35 binding sites upstream |
| 236144 | EW435 4x(ZNF35/IKZF1tar)-SFFV-GFP | Fluorescent reporter encoding GFP under control of SFFV promoter with four ZNF35/IKZF1 binding sites upstream |
| 236145 | EW369 CMV-TO-ZNF35(5-8)-ZNF250(1-4)-deImmunLink-NZF(FLP-IN) | Plasmid encoding the ZNF35/ZNF250 zinc finger array attached by a deimmunized linker to the NZF transcriptional activation domain, under control of CMV promoter with two TetR binding sites |
| 236146 | EW499 CMV-TO-NZF-(GGS)x8[G1W S3D S6T G7L S9L G11M G14N G17D G19V G22W]-FRB(N2093W, T2098L) | Plasmid encoding the NZF transcriptional activation domain attached by a deimmunized linker to FRB with N2093W and T098L mutations, under control of CMV promoter with two TetR binding sites |
| 236147 | EW489 CMV-TO-ZNF35(5-8)-ZNF250(1-4)-(GGS)x8[G2V G4W S6L G7L G10W G11D G13L G17M G19I G22M G23Q S24Q]-FKBP (FLP-IN) | Plasmid encoding the ZNF35/ZNF250 zinc finger array attached by a deimmunized linker to FKBP1A, under control of CMV promoter with two TetR binding sites |
| 236148 | EW411 (UTRN-43-5bp spacer-UTRN-14)-H2B-GFP | Fluorescent reporter encoding H2B-GFP fusion under control of E1b minimal promoter with the UTRN promoter region encompassing 14 to 67 bp upstream of its transcriptional start site |
| 236149 | EW490 CMV-TO-UTRN-14ZF-realdeImmLink-NZF (FLP-IN) | Plasmid encoding the UTRN-14 zinc finger array attached by a deimmunized linker to the NZF transcriptional activation domain, under control of CMV promoter with two TetR binding sites |
| 236150 | EW492 CMV-TO-UTRN-14ZF(N61K,H88F, I91E, K120F)-realdeImmLink-NZF(FLP-IN) | Express mutated UTRN-14 zinc finger array (N62K, H89F, I91E, K121F) attached by a deimmunized linker to the NZF transcriptional activation under control of CMV promoter with two TetR binding sites |
| 236151 | SCN1Aprom(335-406)-H2B-GFP | Fluorescent reporter encoding H2B-GFP fusion under control of E1b minimal promoter with the SCN1a promoter region encompassing 335 to 406 bp upstream of its transcriptional start site |
| 236152 | JPZF1 CMV-TO-SCN1A-377-NZF | Plasmid encoding the SCN1A-377 zinc finger array attached by a GS linker to the NZF transcriptional activation domain, under control of CMV promoter with two TetR binding sites |
| 236153 | JPZF1 CMV-TO-SCN1A-377(G2E, A104K, S109I)-deImmLink-NZF | Express mutated SCN1A-14 zinc finger array (G2E, A104K, S109I) attached by a deimmunized linker to the NZF transcriptional activation domain, under control of CMV promoter with two TetR binding sites |
| 236154 | EW368 CMV-TO-UtroUpZF-deImmunLink-NZF(FLP-IN) | Plasmid encoding the UtroUp zinc finger array attached by a deimmunized linker to the NZF transcriptional activation domain, under control of CMV promoter with two TetR binding sites |
| 236155 | EW418 PGK-IKZF1(2-3)-realDeimmunLink-NZF (FLP-IN) | Plasmid encoding the IKZF1-derived portion of the ZNF35/IKZF1 zinc finger array attached by a deimmunized linker to the NZF transcriptional activation domain, under control of PGK promoter |
| 236156 | EW410 IKZF1tar x8-H2B-GFP | Fuorescent reporter encoding H2B-GFP fusion under control of E1b minimal promoter with eight IKZF1 binding sites upstream |
| 236157 | EW392 CMV-TO-NZF-(GGS)x8-FRB(T2098L) (FLP-IN) | Expresses NZF transcriptional activation domain attached by a GS linker to the mTOR FRB rapamycin binding domain with the T098L mutation, under control of CMV promoter with two TetR binding sites |
| 236158 | EW394 CMV-TO-ZNF35(5-8)-ZNF250(1-4)-(GGS)x8-FKBP (FLP-IN) | Plasmid encoding the ZNF35/ZNF250 zinc finger array attached by a GS linker to FKBP1A, under control of CMV promoter with two TetR binding sites |
| 236159 | EW488 CMV-TO-NZF-(GGS)x8[G1W S3D S6T G7L S9L G11M G14N G17D G19V G22W]-FRB(T2098L) | Express NZF transcriptional activation domain fused to a deimmunized linker and mTOR FRB rapamycin binding domain with the T098L mutation, under control of CMV promoter with two TetR binding sites |
| 236160 | EW172 CMV-TO-UTRN-14ZF-VP64 (FLP-IN) | Plasmid encoding the UTRN-14 zinc finger array attached by a GS linker to the VP64 transcriptional activation domain, under control of CMV promoter with two TetR binding sites |
| 236161 | EW171 CMV-TO-UTRN-43ZF-VP64 (FLP-IN) | Plasmid encoding the UTRN-43 zinc finger array attached by a GS linker to the VP64 transcriptional activation domain, under control of CMV promoter with two TetR binding sites |
| 236162 | SCN1Aprom(1-500)-H2B-GFP | Fluorescent reporter encoding H2B-GFP fusion under control of E1b minimal promoter with the SCN1a promoter region encompassing 500 bp upstream of its transcriptional start site |
| 236163 | JPZF2 CMV-TO-SCN1A-374-NZF | Plasmid encoding the SCN1A-374 zinc finger array attached by a GS linker to the NZF transcriptional activation domain, under control of CMV promoter with two TetR binding sites |
| 236164 | JPZF3 CMV-TO-SCN1A-117-NZF | Plasmid encoding the SCN1A-117 zinc finger array attached by a GS linker to the NZF transcriptional activation domain, under control of CMV promoter with two TetR binding sites |
| 236165 | JPZF4 CMV-TO-SCN1A-383-NZF | Plasmid encoding the SCN1A-383 zinc finger array attached by a GS linker to the NZF transcriptional activation domain, under control of CMV promoter with two TetR binding sites |
| 236166 | JPZF5 CMV-TO-SCN1A-472-NZF | Plasmid encoding the SCN1A-472 zinc finger array attached by a GS linker to the NZF transcriptional activation domain, under control of CMV promoter with two TetR binding sites |
| 236167 | JPZF6 CMV-TO-SCN1A-114-NZF | Plasmid encoding the SCN1A-114 zinc finger array attached by a GS linker to the NZF transcriptional activation domain, under control of CMV promoter with two TetR binding sites |
| 236168 | JPZF7 CMV-TO-SCN1A-140-NZF | Plasmid encoding the SCN1A-140 zinc finger array attached by a GS linker to the NZF transcriptional activation domain, under control of CMV promoter with two TetR binding sites |
| 236169 | JPZF8 CMV-TO-SCN1A-475-NZF | Plasmid encoding the SCN1A-475 zinc finger array attached by a GS linker to the NZF transcriptional activation domain, under control of CMV promoter with two TetR binding sites |
| 236170 | JPZF9 CMV-TO-SCN1A-371-NZF | Plasmid encoding the SCN1A-371 zinc finger array attached by a GS linker to the NZF transcriptional activation domain, under control of CMV promoter with two TetR binding sites |
| 236171 | JPZF10 CMV-TO-SCN1A-372-NZF | Plasmid encoding the SCN1A-372 zinc finger array attached by a GS linker to the NZF transcriptional activation domain, under control of CMV promoter with two TetR binding sites |
| 236172 | EW370 (PB) (ZNF35/ZNF250tarv3-6bp spacers)x4-H2B-BFP insul CMV-TO-ZNF35(5-8)-ZNF250(1-4)-deimlink-NZF | PiggyBac-integrated plasmid containing hygromycin resistance gene, ZNF35/ZNF250-driven BFP, and ZNF35/ZNF250-NZF transcription factor |
| 236173 | EW371 (PB) (ZNF35/ZNF35tar-6bp spacers)x4-H2B-BFP insul CMV-TO-ZNF35(5-8)-ZNF35(5-8)-deimlink-NZF | PiggyBac-integrated plasmid containing hygromycin resistance gene, ZNF35/ZNF35-driven BFP, and ZNF35/ZNF35-NZF transcription factor |
| 236174 | EW372 (PB) (ZNF35/IKZF1tar-6bp spacers)x4-H2B-BFP insul CMV-TO-ZNF35(5-8)-IKZF1(2-4)-deimlink-NZF | PiggyBac-integrated plasmid containing hygromycin resistance gene, ZNF35/IKZF1-driven BFP, and ZNF35/IKZF1-NZF transcription factor (with the IKZF1 portion containing ZFs 2-4 from that protein) |
| 236175 | EW373 (PB) (ZNF35/ZNF250tarv3-6bp spacers)x4-H2B-BFP insul CMV-TO-GFP-deimlink-NZF | PiggyBac-integrated plasmid containing hygromycin resistance gene, ZNF35/ZNF250-driven BFP, and GFP-NZF dummy transcription factor |
| 236176 | EW374 (PB) (ZNF35/ZNF35tar-6bp spacers)x4-H2B-BFP insul CMV-TO-GFP-deimlink-NZF | PiggyBac-integrated plasmid containing hygromycin resistance gene, ZNF35/ZNF35-driven BFP, and GFP-NZF dummy transcription factor |
| 236177 | EW375 (PB) (ZNF35/IKZF1tar-6bp spacers)x4-H2B-BFP insul CMV-TO-GFP-deimlink-NZF | PiggyBac-integrated plasmid containing hygromycin resistance gene, ZNF35/IKZF1-driven BFP, and GFP-NZF dummy transcription factor |