Protocol for auxin-inducible depletion of the RNA-binding protein PTBP1 in mouse embryonic stem cells

Summary Inducible degradation of proteins of interest provides a powerful approach for functional studies. Here, we present a protocol for tightly controlled depletion of the RNA-binding protein PTBP1 in mouse embryonic stem cells (ESCs). We describe steps for establishing an ESC line expressing doxycycline-inducible auxin receptor protein OsTIR1 and tagging endogenous Ptbp1 alleles using CRISPR-Cas9 and homology-directed repair reagents. We then detail procedures for assaying the efficiency of inducible PTBP1 knockdown by immunoblotting. This protocol is adaptable for other protein targets. For complete details on the use and execution of this protocol, please refer to Iannone et al.1


Protocol
Protocol for auxin-inducible depletion of the RNAbinding protein PTBP1 in mouse embryonic stem cells Yaroslav Kainov, 1,2,3 Anna Zhuravskaya, 1,2,3 and Eugene V. Makeyev 1,4, * 1 Centre for Developmental Neurobiology, King's College London, London SE1 1UL, UK 2 These authors contributed equally 3 Technical contact: yaroslav.kainov@kcl.ac.uk; anna.zhuravskaya@kcl.ac.uk 4 Lead contact *Correspondence: eugene.makeyev@kcl.ac.uk https://doi.org/10.1016/j.xpro.2023.102644SUMMARY Inducible degradation of proteins of interest provides a powerful approach for functional studies.Here, we present a protocol for tightly controlled depletion of the RNA-binding protein PTBP1 in mouse embryonic stem cells (ESCs).We describe steps for establishing an ESC line expressing doxycycline-inducible auxin receptor protein OsTIR1 and tagging endogenous Ptbp1 alleles using CRISPR-Cas9 and homology-directed repair reagents.We then detail procedures for assaying the efficiency of inducible PTBP1 knockdown by immunoblotting.This protocol is adaptable for other protein targets.For complete details on the use and execution of this protocol, please refer to Iannone et al. 1

BEFORE YOU BEGIN
The auxin-inducible degron (AID) system provides an efficient approach for rapid depletion of a protein of interest in mammalian cells. 2,3The system relies on a plant-derived ubiquitin ligase component that marks degron peptide-tagged proteins for proteosome-dependent degradation in the presence but not in the absence of the plant hormone auxin.The ability of this method to deplete specific proteins within just a few hours makes it a valuable tool for functional studies.For example, rapid depletion of an RNA-binding protein streamlines the analysis of its direct targets while minimizing any indirect effects on the transcriptome.
Here we present an optimized method for inducible depletion of the RNA-binding protein PTBP1 in mouse embryonic stem cells (ESCs).We first describe the procedures used to prepare media for feeder-free ESC cultures, propagate the parental ESC line A2lox, and retrofit it with a doxycycline-inducible transgene encoding the auxin-inducible ubiquitin ligase component OsTIR1 (Figure 1).We then provide a step-by-step protocol for CRISPR-Cas9 and homology-directed repair (HDR) mediated knock-in of mini-AID (mAID) sequences into the endogenous Ptbp1 loci (Figure 2), as well as validating the knock-in clones and assaying them for inducible PTBP1 depletion (Figure 3).Although our description is focused on the PTBP1 example, we believe that the protocol can be readily adapted for other proteins of interest by selecting appropriate CRISPR-Cas9 and HDR constructs.
This part of the protocol describes routine cultivation of A2lox mouse embryonic stem cells, adapted from Mulas et al. 5 CRITICAL: All cell media and solutions should be pre-warmed prior to use.
1. Defrost a vial of A2lox cells (a mouse ESC line for Cre-mediated knock-in of doxycycline-inducible transgenes 4 ) at 37 C in a water bath.Immediately mix the cell suspension with 1-2 mL of prewarmed DMEM/F12 medium and spin at 260 3 g for 5 min, remove the medium and plate the cells in 5 mL of 2i+LIF to a 6-cm dish pre-coated with 0.1% gelatin (Merck, cat# ES-006-B).
Grow the cells in a humidified incubator at 37 C, 5% CO2.70%-80% of cells should be attached 24 h after plating.
Note: For coating, cover the bottom of a tissue culture (TC) grade 6-cm Nunc dish (Thermo Fisher Scientific, cat# 150318) with 2 mL of 0.1% gelatin and incubate the dish for 10-30 min at 37 C. Then aspirate gelatin, wash a dish with PBS and aspirate PBS prior to seeding cells.Alternatives: To reduce costs, the concentration of FBS used for quenching can be decreased to 4%-5%.
d. Collect the cells by centrifugation at 260 3 g for 5 min, aspirate the supernatant, resuspend the pellet in 4 mL of Neurobasal medium and repeat the centrifugation.e. Resuspend the pellet in 2i+LIF and plate the cells to a fresh gelatin-coated dish at a 1:2-1:6 dilution.Note: ESCs tend to grow as dome-shaped, loosely attached colonies.Adding 1%-2% of FBS to 2i+LIF improves colony attachment. 6We occasionally use this approach to minimize colony loss in ESC cloning experiments (see below).However, FBS may also promote ESC differentiation and should be avoided during routine ESC passaging.

4.
Subclone the open reading frame (ORF) of the auxin receptor F-box protein OsTIR1 into the p2Lox-GFP plasmid (Addgene #34635; Reference4 ) in place of GFP using an appropriate cloning strategy.
5. Passage A2lox cells 2-3 times after taking them from cryostorage, as described above.6.On the day before transfection, add 1 mg/mL of doxycycline (Dox; Sigma, cat# D9891-1G) to the A2lox culture to activate the expression of the Cre recombinase for 16 h.
CRITICAL: This step is essential for successful knock-in of the OsTIR1 transgene.
7. Next morning, trypsinize the culture, quench trypsin, and count the cells using hemocytometer.Spin down 0.75-1310 6 cells, discard the supernatant, and resuspend the pellet in 4 mL of 2i+LIF.
Place the suspension into a 6-cm sterile bacterial dish (Corning, cat# BP53-06).Conversely, and mAID-tagged PTBP1 is efficiently depleted in response to the Dox and IAA treatment in a biallelically tagged Ptbp1-mAID clone.As expected, mAID-tagged PTBP1 protein migrates slower than its wild-type counterpart (64.7 kDa vs. 56.9kDa, respectively).
Note: Bacterial 6-cm dishes are used to prevent cell attachment during transfection in suspension.
8. Transfect the cells by adding 1 mg of pML33 mixed with 3 mL of Lipofectamine 2000 (Thermo Fisher Scientific, cat# 11668019) and 100 mL of Opti-MEM I (Thermo Fisher Scientific, cat# 31985070) and incubating the suspension in a TC incubator for 2 h at 37 C (Figure 1A).
Note: Use p2Lox-GFP and mock-transfected cells as a positive and negative control, respectively.
9. Collect the cells by centrifugation for 5 min at 260 3 g, aspirate the supernatant, resuspend the pellet in 2 mL in 2i+LIF, and plate duplicated 100, 200 and 500 mL aliquots to individual wells of a gelatin-coated 6-well TC plate in the final volume of 2 mL of 2i+LIF per well.10.After 24-48 h in a TC incubator: a. Replace the medium with 2 mL/well of 2i+LIF containing 350 mg/mL of G418/geneticin (Thermo Fisher Scientific, cat# 10131019).b.Propagate the cells for 10-15 days with regular medium changes (every 1-3 days depending on the extent of cell death) to allow G418-resistant cells to form colonies.
Note: Colonies ready for picking should be visible without a microscope in cultures transfected with the pML33 and p2Lox-GFP plasmids but not in mock-transfected wells.
Note: If colony detachment becomes a problem, add 1% of FBS to the medium 4-5 days prior to colony picking.

Note:
We recommend performing a killing curve assay to confirm the efficiency of G418 before beginning the cloning experiment.
11. Just before picking the colonies, pre-coat a flat-bottom 96-well TC-grade plate with gelatin as described above and add 200 mL of 2i+LIF with 2% FBS and 100 mg/mL of G418 per well.Also prepare a U-bottom 96-well plate (Greiner Bio-One, cat# 650180) with 50-mL aliquots of 0.05% Trypsin-EDTA per well.12. Clean a stereomicroscope installed in a laminar flow cabinet with 70% ethanol (Figure 1B).13.Find a well with well-separated G418-resistant colonies and dilute the conditioned medium in this well with 2 mL of PBS.14. Pick the colonies using the stereomicroscope and a 20-mL automatic pipette equipped with sterile filter tips (Figure 1B).Gently dislodge a colony by pushing it with a tip, carefully suck it inside the tip, and deposit into a U-bottom well containing a 50-mL aliquot of 0.05% Trypsin-EDTA.Change the tip.Repeat.15.Once all colonies are transferred to the U-bottom plate, incubate the plate at 37 C for 5-10 min.16.Pipette the solution up and down with a multichannel pipette to dissociate the colonies and transfer the entire volume to the flat-bottom 96-well plate with 200 mL of 2i+LIF with 2% FBS and 350 mg/mL G418.17.Next morning, change the medium to 2i+LIF with 100 mg/mL of G418 but without FBS.18.When the cells in the 96-well plate reach 60%-70% confluence (typically in 2-4 days) trypsinize the cells and passage them through the 24-well plate, 6-well plate, and finally the 6-cm dish format.
Note: In our experience, nearly all G418-resistant clones in the A2lox system express the transgene in a Dox-inducible manner.However, we recommend screening 6-12 clones and selecting the clones that grow well, form healthy-looking colonies, and express readily detectable amounts of OsTIR1 mRNA in Dox-but not control-treated samples (see below).
19. Cryopreserve the clones from 6-cm dish cultures using the procedure described in the "propagating ESCs in serum-free 2i+LIF medium" section.We typically prepare 6-83 0.5-mL aliquots from a 60%-80% confluent 6-cm dish and use them to revive the cells in the 6-cm dish format.
Pause point: Clones can be cryopreserved as 1-2 aliquots at the 24-well or the 6-well passaging step and expanded when convenient.20.To analyze OsTIR1 expression in the resultant TRE-OsTIR1 clones (TRE refers to the tetracycline/ doxycycline inducible promoter driving OsTIR1 expression in this system): a. Plate 0.5-1310 5 cells per well of a 12-well plate in 1 mL of 2i+LIF supplemented with 1 mg/mL of Dox. b.After at least 12 h, purify total RNA using the EZ-10 DNAaway RNA Miniprep Kit as recommended by the manufacturer.
Alternatives: Other RNA purification methods can be used as an alternative to the EZ-10 DNAaway RNA Miniprep Kit.For example, we have a positive experience with Monarch Total RNA Miniprep Kit (NEB, cat# T2010S) and PureLink RNA Mini Kit (Thermo Fisher Scientific, cat# 12183018A).
c. Generate cDNA using a reverse transcriptase (RT) kit of your choice (we use the SuperScript IV kit with random decamer primers as recommended by the manufacturer).d.Perform qPCR with OsTIR1_F/OsTIR1_R primers.Use Cnot4 as a housekeeping control for qPCR signal normalization.See Table 1 for primer sequences.
Note: We use the following amplification conditions:

Protocol
Note: Mix 1003 N2 well by gently pipetting the final mixture up and down, aliquot by 1 mL, and store at -80 C for up to one year.

Note:
The medium can be stored for up to one month at +4 C.
Note: Use freshly prepared 100 mM b-mercaptoethanol.Dilute 100 mL of b-mercaptoethanol with 14.1 mL of TC-grade sterile distilled H 2 O. Sterilize through a 0.2-mm filter and store at 4 C for up to 1 month.

STEP-BY-STEP METHOD DETAILS
This protocol describes tagging of both alleles of the Ptbp1 gene with C-terminal mini-AID (mAID) sequences in TRE-OsTIR1 ESCs introduced above.We further outline the procedure for assaying inducible degradation of mAID-tagged PTBP1.In short, we co-transfect cells with a Ptbp1-specific CRISPR-Cas9 construct and two Ptbp1-specific HDR constructs with different selection markers.We provide HDR constructs with three markers to choose from: HygR, BSD and PuroR (conferring mammalian cell resistance against hygromycin B, blasticidin S and puromycin, respectively; Addgene ## 206997, 206998, 206999).However, we tend to obtain more biallelically tagged TRE-OsTIR1 clones using the HygR and BSD combination and describe this selection approach below.Once the Ptbp1-mAID knock-in clones (with the TRE-OsTIR1, Ptbp1 mAID/mAID genotype) are established and validated, they are treated with Dox and auxin (indole-3-acetic acid, or IAA), and the expression of OsTIR1 and mAID-tagged PTBP1 (PTBP1-mAID) is assayed by RT-qPCR and immunoblotting.

Timing: 3-4 weeks
This part of the protocol describes biallelic tagging of the mouse Ptbp1 gene with auxin-inducible degron sequences.The TRE-OsTIR1 cells used for this purpose are described in the before you begin section.Protocol Note: In the example described in this section, the mAID tag is added to the C-terminus of the protein of interest.The knock-in strategy should be modified accordingly if N-terminal tagging is required.
2. Generate HDR constructs for in-frame C-terminal mAID tagging of your gene of interest with 2 different markers.

Note:
We provide three such constructs for Ptbp1 encoding the HygR, BSD, and PuroR eukaryotic selection markers (Addgene ##206997, 206998, 206999) and recommend using the HygR and BSD combination for targeting the TRE-OsTIR1 cells.To build these constructs, Ptbp1-specific left and right homology arms were amplified from mouse genomic DNA using the Ptbp1_HAL_F/Ptbp1_HAL_R and Ptbp1_HAR_F/Ptbp1_HAR_R primer pairs (Table 1), respectively.The left and the right arms were then cloned into pML42 (a HygR-encoding vector) and pML49 (a PuroR-encoding vector) at the SacI-BamHI and the BstBI-AvrII sites, respectively, producing the pML58 and pML60 HDR constructs. 1 To prepare the BSD-containing HDR construct pML646, pML58 was cut with NheI-Acc65I and HygR was replaced by a BSD-containing fragment amplified from pLenti6-LacZ (Invitrogen) using the BSD_F/BSD_R primer pair (Table 1).For best results, we recommend keeping the length of the homology arms >500 bp.
Note: Use mock-transfected cells as a negative control.
6. Collect the cells by centrifugation for 5 min at 200 3 g.

7.
Resuspend the pellet in 2 mL of 2i+LIF, and plate duplicated 100, 200 and 500 mL aliquots to individual wells of a gelatin-coated 6-well TC plate in the final volume of 2 mL of 2i+LIF supplemented with 0.5 mM Alt-R HDR Enhancer V2 (IDT, cat# 10007910).8.After 24-48 h in a TC incubator, replace the medium with 2 mL/well of 2i+LIF containing 150 mg/mL of hygromycin B and 8 mg/mL of blasticidin S. 9. Propagate the cells for an additional 10-15 days with regular medium changes (every 1-3 days depending on the extent of cell death) to allow antibiotic-resistant cells to form colonies.

Note:
We recommend performing killing curve assays to confirm the efficiency of hygromycin B and blasticidin S before beginning the cloning experiment.
Note: Although mAID knock-in TRE-OsTIR1 cells (e.g., Ptbp1-mAID) remain resistant to G418, we do not use this antibiotic when selecting hygromycin B and blasticidin S resistant clones to avoid excessive cell stress.G418 can be added to the medium at 100 mg/mL once the mAID knock-in line has been established.
10. Pick and dissociate the colonies as described in the "knocking in the OsTIR1 transgene into A2lox ESC genome" section.Transfer the entire volume to a gelatinized TC-grade flat-bottom 96-well plate containing 200 mL of 2i+LIF with 2% FBS and 150 mg/mL of hygromycin B and 8 mg/mL of blasticidin S per well.
Note: Colonies ready for picking should be visible without a microscope in cultures transfected with the CRISPR-Cas9 and HDR plasmid mixture but not in the mock-transfected control (Figures 2B-2D).
Note: If colony detachment becomes a problem, add 1% of FBS to the medium 4-5 days prior to colony picking.
11. Next morning, change the medium to 2i+LIF without FBS and with 100 mg/mL of hygromycin B and 5 mg/mL of blasticidin S per well.12.When the wells reach 60%-70% confluence (typically in 2-4 days): a. Trypsinize the cells.b.Quench with 200 mL of 2i+LIF with 2% FBS.c. Transfer half of the volume to a fresh gelatinized TC-grade flat-bottom 96-well plate containing 100 mL of 2i+LIF with 2% FBS and 100 mg/mL of hygromycin B and 5 mg/mL of blasticidin S per well.
Note: Use the remaining half of the volume for DNA purification and PCR genotyping as soon as possible (see below).
13. Based on the PCR genotyping results (see below), select correct clones and passage them through the 24-well plate, 6-well plate, and 6-cm dish steps in 2i+LIF without FBS and with 100 mg/mL of hygromycin B and 5 mg/mL of blasticidin S. 14. Cryopreserve the clones from 6-cm dish cultures using the procedure described in the "propagating ESCs in 2i medium with LIF" section.
Note: We typically prepare 6-8 3 0.5-mL aliquots from a 60%-80% confluent 6-cm dish and use them to revive the cells in the 6-cm dish format.
Pause point: Clones can be cryopreserved as 1-2 aliquots at the 24-well or the 6-well passaging step and expanded when convenient.

Timing: 1 week
This part of the protocol describes molecular characterization of the mAID-tagged ESC clones.CRITICAL: Perform genotyping as soon as possible to reduce the workload associated with passaging of numerous clones.
15. Centrifuge the 96-well plate with remaining half of trypsinized cells from step 12 of the previous section using a 96-well plate rotor at 1500 3 g for 10 min.

Note:
We typically centrifuge cells in the flat-bottom plates where they grew.However, to analyze <60% confluent cultures, cell suspensions should be transferred to 96-well PCR plates prior to centrifugation to avoid losing the pellets.
16. Remove the supernatants using a multichannel pipette and extract the DNA from the cell pellets using PCRBIO Rapid Extract PCR Kit, as recommended by the manufacturer.
Alternatives: Resuspend the cell pellets in the smaller volume of the final PCRBIO Rapid Extract mix (down to 30-40 mL) and incubate the plate for 5 min at 75 C and then for 10 min at 95 C.

2 .
Passage the cells every 2-3 days: a. Aspirate the medium, and rinse the plate with 2 mL PBS.b.Add 1 mL of 0.05% Trypsin-EDTA (Thermo Fisher Scientific, cat# 15400054 diluted 10 times with PBS), and incubate the plate for up to 10 min at 37 C. Note: Trypsinize cells at 37 C until R90% of the cells have detached.Observe the cells under the microscope to control detachment and avoid possible over-trypsinization.c.Quench trypsin with 4 mL of a 1:3 (v/v) mixture of ESC-grade FBS (Thermo Fisher Scientific, cat# SH30070.03E) and DMEM/F12 medium and pipette the suspension up and down a few times to dissociate cell clumps.

Figure 1 .
Figure 1.Preparing TRE-OsTIR1 ESCs (A) Cre/Lox recombination-mediated monoallelic knock-in of the OsTIR1 transgene into the A2lox mouse ESC line.See 4 for a more detailed description of A2lox system.(B) The setup we use to pick ESC colonies.

3 .
To cryopreserve A2lox cells: a. Remove the supernatant after the FBS quenching step and resuspend the cells in a required amount of the Recovery medium (Thermo Fisher Scientific, cat# 11560446).b.Prepare 0.5 mL aliquots in cryovials, place the aliquots into a Mr. Frosty freezing container (Thermo Fisher Scientific, cat# 5100-0001) at 20 C-25 C, and store the container at À80 C for 1-2 days.c. Transfer the vials to liquid nitrogen for long-term storage.Knocking in the OsTIR1 transgene into A2lox ESC genomeTiming: 3-4 weeksThis part of the protocol describes Cre-mediated knock-in of a single copy of the OsTIR1 transgene into a lox2272/loxP-containing locus of the A2lox ES cell line developed in the Kyba lab.4

Figure 2 .
Figure 2. Biallelic tagging of the Ptbp1 gene with mAID sequences (A) To knock in mAID into both allelic Ptbp1 loci, TRE-OsTIR1 ESCs are co-transfected with an appropriate CRISPR-Cas9 plasmid and two homology-directed repair (HDR) constructs encoding the HygR and BSD selection markers.Ptbp1 HAL and Ptbp1 HAR and the left and right homology arms, respectively.(B-D) Examples of antibiotic-resistant ESC colonies formed in the experiment depicted in (A).(B) Undifferentiated colonies resistant to both hygromycin B and blasticidin S formed in a successfully co-transfected TRE-OsTIR1 ESC culture.(C) Another field containing an undifferentiated (filled arrowhead) colony suitable for picking and a differentiated colony (open arrowhead) that should not be used for further experiments.(D) No antibiotic-resistant colonies are detectable in a mock-transfected TRE-OsTIR1 ESC culture.Scale bars in (B-D), 1 mm.

Figure 3 .
Figure 3. Genotyping mAID knock-in ESC clones and assaying inducible degradation of mAID-tagged PTBP1 (A) PCR genotyping strategy used to analyze mAID knock-in results.Top, the wild-type Ptbp1 locus.Bottom, successfully targeted Ptbp1 locus.See Table 1 for primer sequences.(B) Initial PCR screening of hygromycin B-and blasticidin S-resistant clones with primers introduced in (A).Arrowheads, clones 13 and 21 give rise to the mAID-specific but not the wild type-specific PCR product (expected product sizes are 775 bp and 1507 bp, respectively) indicating successful tagging of both Ptbp1 alleles.(C) More detailed genotyping of the parental TRE-OsTIR1 ESC line and one of its Ptbp1-mAID clones with Ptbp1 biallelically tagged with mAID using primer pairs indicated.The expected product sizes are: F1/R1, 385 bp; F1/R3, 1507 bp (wild type) and 3237 bp (mAID cassette knock-in; not detectable under the PCR conditions used); F1/R2, 775 bp; Fh/R3, 1298 bp; and Fb/R3, 1325 bp.(D) Immunoblot analysis showing stable expression of the wild-type PTBP1 in the parental TRE-OsTIR1 cells treated with Dox and IAA for 24 h.Conversely, and mAID-tagged PTBP1 is efficiently depleted in response to the Dox and IAA treatment in a biallelically tagged Ptbp1-mAID clone.As expected, mAID-tagged PTBP1 protein migrates slower than its wild-type counterpart (64.7 kDa vs. 56.9kDa, respectively).

Table 1 .
Oligonucleotides used in this protocol 2 O 6.5 mL STAR Protocols 4, 102644, December 15, 2023 Note: Prepare 100 mg/mL apo-transferrin by dissolving the contents of a 100 mg vial in 1 mL DMEM/F12 immediately before making 1003 N2.Use the 1003 N2 solution to wash the vial and improve apo-transferrin recovery.Make sure that apo-transferrin is completely dissolved before aliquoting 1003 N2. mL STAR Protocols 4, 102644, December 15, 2023

Table 1 )
1. Generate a CRISPR-Cas9 construct targeting the junction between the open reading frame and the 3 0 UTR in your gene of interest using an appropriate gRNA design tool (e.g., https://www.benchling.com).into pX330 8 cut with BbsI.