A Single-step Generation of AlissAID-based Conditional Knockdown Strains Using Nanobody that Targets GFP or mCherry in Budding Yeast

The Auxin-inducible degron (AID) system is a genetic tool that induces rapid target protein depletion in an auxin-dependent manner. Recently, two advanced AID systems—the super-sensitive AID and AID 2—were developed using an improved pair of synthetic auxins and mutated TIR1 proteins. In these AID systems, a nanomolar concentration of synthetic auxins is sufficient as a degradation inducer for target proteins. However, despite these advancements, AID systems still require the fusion of an AID tag to the target protein for degradation, potentially affecting its function and stability. To address this limitation, we developed an affinity linker–based super-sensitive AID (AlissAID) system using a single peptide antibody known as a nanobody. In this system, the degradation of GFP- or mCherry-tagged target proteins is induced in a synthetic auxin (5-Ad-IAA)–dependent manner. Here, we introduce a simple method for generating AlissAID strains targeting GFP or mCherry fusion proteins in budding yeasts. Key features • AlissAID system enables efficient degradation of the GFP or mCherry fusion proteins in a 5-Ad-IAA–depending manner. • Transforming the pAlissAID plasmids into strains with GFP- or mCherry- tagged proteins.

This protocol is used in: PLOS Genetics (2023), DOI: 10.1371/journal.pgen.1010731 The Auxin-inducible degron (AID) system is a genetic tool that induces rapid target protein depletion in an auxindependent manner.Recently, two advanced AID systems-the super-sensitive AID and AID 2-were developed using an improved pair of synthetic auxins and mutated TIR1 proteins.In these AID systems, a nanomolar concentration of synthetic auxins is sufficient as a degradation inducer for target proteins.However, despite these advancements, AID systems still require the fusion of an AID tag to the target protein for degradation, potentially affecting its function and stability.To address this limitation, we developed an affinity linker-based super-sensitive AID (AlissAID) system using a single peptide antibody known as a nanobody.In this system, the degradation of GFP-or mCherry-tagged target proteins is induced in a synthetic auxin (5-Ad-IAA)-dependent manner.Here, we introduce a simple method for generating AlissAID strains targeting GFP or mCherry fusion proteins in budding yeasts.5

5-Ad-IAA
Dissolve in DMSO at a concentration of 5 mM and store at -30 °C.When it is used for liquid medium, add 1/1,000 of the amount of 5 mM 5-Ad-IAA to the medium.When it is used for plate media, autoclaved media should be cooled to approximately 50 °C before adding 5-Ad-IAA.5-Ad-IAA containing plates can be stocked at 4°C, hidden from direct light, for at least one month.

A. Preparation of GFP-or mCherry-tagged strains
Prepare strains in which target proteins are tagged with GFP or mCherry.It is easy to add fluorescent tags to an ORF in its chromosomal location through homologous recombination in budding yeast [10].Alternatively, the appropriate strains were selected from the GFP Clone Collection [10].In this paper, we used W303-1a strains that were tagged at the C-terminus of ASK1 on the genome and selected with HIS3 using the method previously reported [9].
Note: Although we have not tested the protein degradation of N-terminal-tagged proteins, in principle, the AlissAID system would work on N-terminal tagged proteins.

B. Transformation of the pAlissAID plasmid
Note: Pay attention to the selection marker; in the case of the GFP Clone Collection, the GFP sequence is inserted at the C-terminus of the target gene using HIS3 as the selection marker.Therefore, we used the pAlissAID plasmids encoding TRP1, LEU2, and URA3 (Figure 2).All these pAlissAID plasmids (HIS3, TRP1, LEU2, and URA3 encoding) are available from NBRP.Day 1 1.Grow yeast cells overnight in 5 mL of YPD medium at 30 °C with shaking at 230 rpm.

Day 2
1. Dilute the cells to OD600 = 0.3 and regrow the cells in 5 mL of YPD medium at 30 °C with shaking at 230 rpm for 3-5 h.11.Centrifuge (20,000× g, 30 sec, room temperature) and discard the supernatant.12. Resuspend the pellet in the remaining supernatant (at least 50 μL is needed) and plate the cells onto an SD plate medium (lacking histidine, leucine, tryptophan, or uracil).13.Incubate at 30 °C for 2-3 days.dilution spotting of control and AlissAID strains on SD medium plate with or without 5.0 µM 5-Ad-IAA.10-fold serial dilutions were made from the cells with OD600 = 0.3 and spotted into the plate.The strain that expresses Ask1 instead of Ask1-GFP and contains pAlissAID plasmid is used as a control.Cells were grown for 48 h at 30 ℃.

When cells have grown to approximately
OD600 = 1.0, centrifuge them (1,000× g, 5 min, approximately 6 Published: Jun 20, 2024 25 °C) and discard the supernatant.3. Suspend cells in 1 mL of sterilized water and transfer to a sterilized 1.5 mL tube.4. Centrifuge (20,000× g, 30 s, 25 °C) and discard the supernatant.5. Resuspend in 1 mL of 0.1 M LiOAc.6.After centrifugation (20,000× g, 30 s, 25 °C), discard the supernatant and centrifuge again to completely remove the supernatant.7. Denature the ssDNA at 90 °C for approximately 5 min and cool on ice.8. Add the following solutions and mix using a vortex.2.0 mg/mL ssDNA, 25 μL pAlissAID plasmid, 1 μg sterilized water, up to 50 μL 9. Add 240 μL of 50% w/v PEG and 36 μL of 1 M LiOAc.Vortex the mixture and incubate in a heat block at 30 °C for 30 min.10.Add 40 μL of DMSO.Vortex the mixture and incubate in a heat block at 42 °C for 20 min.

Figure 3 . 8 Published
Figure 3. Degradation of GFP fusion proteins in AlissAID strains.(A) AlissAID strains were generated by the transformation of pAlissAID plasmids with a different selection marker (TRP1, LEU2, or URA3) into the Ask1-GFP strain.(B) Microscopic observations of fluorescent signals of Ask1-GFP in AlissAID strains.Cells were treated with or without 5.0 µM 5-Ad-IAA for 3 h at 30 ℃. Scale bar, 10 µm.(C) Serial

Figure 4 .
Figure 4. Degradation of mCherry fusion proteins in AlissAID strains.(A) AlissAID strains were generated by the transformation of pAlissAID plasmids with a different selection marker (TRP1, LEU2, or URA3) into the Ask1-mCherry strain.(B) Microscopic observations of fluorescent Ask1-mCherry signals in AlissAID strains.Cells were treated with or without 5.0 µM of 5-Ad-IAA for 3 h at 30 ℃. Scale bar, 10 µm.(C) Serial dilution spotting of control and AlissAID strains on SD medium plate with or without 5.0 µM 5-Ad-IAA.10-fold serial dilutions were made from the cells with OD600 = 0.3 and spotted into the plate.The strain that expresses Ask1 instead of Ask1-mCherry and contains pAlissAID plasmid is used as a control.Cells were grown for 48 h at 30 ℃.