Protocol to target a promoter region in human embryonic kidney cells using the CRISPR-dCas9 system for single-locus proteomics

Summary The unbiased identification of less-abundant transcription factors, which direct the expression of a target gene, is technically challenging. Here, we present a protocol to analyze the locus-specific chromatin-regulating proteome using in situ capture of chromatin interactions by an inactive Cas9 (dCas9). We describe steps for designing guide RNAs and transfection, followed by precipitation of chromatin and associated proteins. In the last step, we describe the elution of DNA and proteins for PCR and mass spectrometric analysis, respectively. For complete details on the use and execution of this protocol, please refer to Alkhayer et al.1

4. Ligate the plasmid and oligo pairs.a. Pipette 50 ng of the digested vector, 2 mL of the annealed and diluted oligos, 2 mL 103 DNA Ligase Buffer, 1 mL T4 Ligase, and nuclease-free water to 20 mL in a 1.5 mL reaction tube.b.Incubate the ligation reaction for 1 h at 20 C-22 C. Transformation Timing: 2.5 days 5. Transform the plasmid into competent cells.
a. Thaw E.coli XL1-blue competent bacteria on ice for about 20 min.b.Add 8 mL of the ligation mixture to 100 mL of the bacteria in a 1.5 mL reaction tube.c.Gently mix by hand and place back on ice for 30 min.d.Heat shock the competent cells with the ligation mixture by exposing them to 42 C for 90 s. e. Cool the mixture on ice for 2 min.f.Add 500 mL of antibiotic-free LB medium to the mixture.g.Incubate for 2 h at 37 C with shaking at 700 rpm.h.Centrifuge the mixture at 300 3 g for 2 min at 20 C-22 C. i. Discard 500 mL of the supernatant.j.Gently resuspend the pellet in the remaining medium.k.Inoculate an LB agar plate containing Kanamycin (50 mg/mL).l.Incubate the plate overnight at 37 C for 16 h.m.Store the plate at 4 C before you proceed to the next step.n.Pick individual colonies and inoculate in 4 mL LB medium containing Kanamycin (50 mg/mL).

Blocking of Sepharose A and G
Timing: 1 day 6.Block Sepharose beads A and G. a. Mix 5 mL of protein A agarose and 5 mL of protein G agarose in a 50 mL falcon containing 15 mL Lysis Buffer II with freshly added 13 protease inhibitor cocktail for washing.b.Centrifuge for 5 min at 1,200 3 g at 4 C and discard the supernatant.c.Repeat this step one more time.d.Resuspend the washed beads in 15 mL Lysis Buffer II with freshly added 13 protease inhibitor cocktail containing 1 g/L BSA and 0.4 g/L sonicated salmon sperm DNA.e. Incubate the beads for 16 h at 4 C on a roller.f.Centrifuge the blocked beads and discard a part of the supernatant (buffer) to have a 50% slurry, meaning if the volume of the beads after centrifugation is 7 mL, leave 7 mL of the buffer and discard the rest.g.Aliquot the blocked beads into a 5 mL reaction tube.h.Store the beads at 4 C for up to a year.

KEY RESOURCES TABLE
Companies are indicated, however, reagents that are available from other sources can also be used, provided they are free of DNA contamination and recommended for cell culture.

STEP-BY-STEP METHOD DETAILS
Cell culture transfection for the ChIP assay

Timing: 3 days
This step describes the transfection of HEK293 cells with dCas9 and gRNA vectors (designed in the ''before you begin'' section) that target the locus of interest (here: MICA promoter).
Note: Human embryonic kidney cells (HEK293) are used in the following steps.
Note: This protocol provides the volumes for a single 15 cm culture dish for each condition.10 3 15 cm dishes were used per condition to obtain sufficient cell numbers for mass spectrometry analysis.
1. Transfect cells with dCas9 and gRNA vectors.a. Thaw the cells and culture them in DMEM medium containing 10% FBS and 1% penicillin/ streptomycin at 37 C with 5% CO 2 .b. Passage the cells twice to three times per week and use them when the passage number is between two and five.c.Seed 5-6 3 10 6 cells in a 15 cm cell culture dish containing 20 mL DMEM medium with 10% FBS and 1% penicillin/streptomycin 20 h before transfection.d. 3 h prior to transfection, replace the medium with penicillin/streptomycin-free DMEM with 10% FBS.

Precipitation of chromatin via dCas9
Timing: 1.5 days This section describes the dCas9-mediated precipitation of the chromatin followed by shearing and subsequent incubation with beads.
2. Prepare native chromatin.a. Fix cells by adding 2 mL formaldehyde (10% stock) to 18 mL of medium for a final concentration of 1%.b.Gently swirl the plate.

Note:
The color of the medium will change from pink to yellow for a few minutes.c.Incubate for 10 min at 20 C-22 C under a chemical fume hood.d.Quench the formaldehyde by adding 1 M glycine to a final concentration of 125 mM.e. Mix by gentle swirling.f.Incubate for 5 min at 20 C-22 C. g.Discard the supernatant and wash the cells twice with cold DPBS.h.Scrap the cells with a rubber policeman, harvest with a 1000 mL pipette tip, and transfer to a 15 mL falcon.i. Centrifuge the cells at 1200 3 g for 5 min at 4 C. j.Discard the supernatant.
Note: In this step, the cell pellet can be stored at -80 C for up to a year.k.Gently but thoroughly resuspend the pellet in 2 mL Lysis Buffer I (add fresh protease inhibitor cocktail 1:1,000) using a 1000 mL pipette tip to obtain a homogenous suspension.l.Incubate for 20 min on ice.
Note: This step is to disrupt the cellular membrane and remove the cytoplasmic components.
m. Centrifuge at 1,200 3 g for 5 min at 4 C. n.Discard the supernatant.o.Gently but thoroughly resuspend the nuclei in 2 mL Lysis Buffer II (add fresh protease inhibitor cocktail 1:1,000).p. Incubate on ice for 10 min.q.Ensure that the suspension is homogeneous and carefully transfer 1 mL each into two new 15 mL falcons with a 1,000 mL pipette tip.
CRITICAL: Avoid the formation of air bubbles.It is critical for the chromatin sonication step to prevent protein denaturation at the surface and loss of chromatin in the bubbles, which may affect the DNA/protein complex required for the precipitation step.

Shear the chromatin.
CRITICAL: For the sonication step, it is essential to keep the pellet cool, which can be achieved by using a sonication system, here the Benchtop Tube Cooler, Active Motif, no.53077.
Note: Always store the 15 mL Benchtop Tube Cooler at À20 C and remove it before use.It can be stored at À80 C but for no longer than 30 min.
Note: To avoid freezing the sample, do not place the sample in the cooler until you are ready to sonicate it directly.

Note:
The setting of the sonication step must be defined for each cell line and sonication device.
a. Set the sonication device according to the cell line (here: HEK293 cell line) as follows: in total 52 pulses of 1 s pulse with 4 s pause at 20% amplitude.b.Fill the 15 mL Benchtop Tube Cooler with 1 mL NaCl (5 M) using a 1000 mL pipette tip.c.Place the falcon in the cooler and directly into the sonicator.

CRITICAL:
The 15 mL falcon should be positioned in the center of the cooler without touching the edge.CRITICAL: Try to preserve the epitopes by sonicating the samples as weakly and briefly as possible.Otherwise, indirectly bound proteins can easily be lost during the procedure.CRITICAL: To avoid foaming of the sample, immerse the tip of the sonicator at 60-80% of the maximum depth.In the case of foaming, the epitope will be affected by oxidation and the shear efficiency will be drastically reduced.b.To determine the chromatin shear efficiency, transfer approximately 10 mL to a new reaction tube.c.Add 20 mg Proteinase K, 20 mg RNase A, and 100 mL mineral oil.d.Mix well by vortexing.e. Incubate for 16 h at 65 C. f.Mix 10 mL of the sample with loading buffer and load onto a 1% agarose gel containing GelRed.g.Run the gel at a maximum voltage of 80 V and check the size of the DNA fragments.
Note: DNA fragments should be between 500 bp and 700 bp.
CRITICAL: The appearance of the pellet can provide information about the sonication efficacy; if the pellet is large and white, it means that the sonication was insufficient.If the pellet is small and transparent with dark speckles, the number of pulses or the amplitude, or both, must be reduced to conserve the epitope.

Prepare the blocked beads for incubation with chromatin.
a. Use a 50% slurry of blocked beads and incubate with a non-specific antibody such as rabbit IgG antibody.b.Add 10 mg IgG per 100 mL of beads.c.Incubate at 4 C for 30 min on a roller.CRITICAL: Prepare enough blocked beads for all samples, 100 mL of the 50% slurry beads blocked with IgG for the 1 mL of the soluble chromatin.In total 2 mL of the beads for each 20 mL of the soluble chromatin.d.Centrifuge the blocked beads at 300 3 g for 1 min at 4 C. e. Carefully aspirate the supernatant.f.Wash the blocked beads twice with Lysis Buffer II (with protease inhibitor cocktail 1:1,000).g.Centrifuge for 1 min at 300 3 g at 4 C. h.Discard the supernatant.i. Add sufficient buffer to achieve 50% slurry of the beads.j.Repeat steps f to i. CRITICAL: Gently handle the beads, taking care not to disrupt them during centrifugation and aspiration of the supernatant; you can use a vacuum system (QIAvac 24 Plus, QIAGEN) connected to a needle to aspirate the supernatant.

Timing: 2.5 days
In the following section, the precipitated DNA and proteins are isolated and prepared for PCR and mass spectrometric analysis, respectively.6. Immunoprecipitate the chromatin (carry this step out in parallel with step 12).a.At this time, collect an aliquot of soluble chromatin equal to 1% of the volume used for immunoprecipitation (IP) and store it at 4 C; this is referred to as the input sample.
Note: In this protocol, 3 mL of soluble chromatin is the required volume.
b. Add 4 mg of IgG antibody to 300 mL of soluble chromatin (for the IP control).c.Add 100-120 mg of Cas9 antibody to the remaining volume of soluble chromatin (approximately 20 mL).d.Incubate the samples for 16 h at 4 C on a roller.
Note: Store the input samples at 4 C for later use.e. Transfer 300 mL of the Cas9-IP into a new reaction tube to proceed with the qPCR assay and confirm precipitation of the target locus.The rest of the Cas9-IP sample is for mass spectrometry analysis.f.For each IP, add 50 mL of the A + G blocked beads slurry (from blocking of Sepharose beads A and G) and rotate for 1 h at 4 C.
CRITICAL: Before using the A + G blocked beads slurry, gently mix to obtain a homogeneous suspension.Try to mix gently by inverting the reaction tube before pipetting each sample.
Note: To remove the 50 mL of bead suspension, use the 200 mL pipette tips and cut off the end with clean scissors.This will make it easier to pipette the 50% bead suspension.
7. Perform a qPCR to detect the target locus (here MICA) using the following 3 samples: a. input sample, b. chromatin precipitated with Cas9 antibody (Cas9-IP), and c. chromatin precipitated with control antibody (IgG-IP).
8. Wash the beads.a. Centrifuge the IP samples at 300 CRITICAL: Keep the samples on ice for steps a to m.
CRITICAL: Handle the beads with care and aspirate the supernatant without disturbing the beads.
Note: For bead washing steps, you can use a vacuum system (QIAvac 24 Plus, QIAGEN) that you connect to a special needle to aspirate the supernatant.9. Elute immunoprecipitates from the beads.
Note: For this step, prepare 400 mL of freshly prepared elution buffer for each sample (also include the input samples in this step).
d. Transfer the sheared chromatin to a new 1.5 mL reaction tube.e. Centrifuge at 13,000 3 g for 15 min at 4 C. Note: This is the soluble chromatin fraction.f.Collect 2 mL of soluble chromatin per 15 cm dish.Note: In this study, a total of 20 mL per condition (10 3 15 cm dishes per condition) was used.4.Determine shearing efficiency.a.Pool the soluble chromatin from twenty 1.5 mL reaction tubes into two 15 mL falcons for the next steps.
k.For chromatin pre-clearing, add 2 mL of the 50% slurry of the beads (blocked with IgG antibody) to 20 mL of the soluble chromatin.l.Incubate for 45 min at 4 C on a roller.m.Centrifuge at 1,200 3 g for 5 min at 4 C. n.Transfer the pre-cleared chromatin to a new falcon tube.o.Combine the two 15 mL falcon tubes of the soluble chromatin in one 50 mL falcon tube.
a. Incubate the beads for each sample with 200 mL of elution buffer for 15 min at 20 C-22 C with vigorous shaking (1,200 rpm).b.Centrifuge the samples at high speed (17,000 3 g) for 2 min at 20 C-22 C. c. Carefully transfer the supernatant to a new reaction tube.d.Repeat steps a and b.Then combine the supernatants.e. Add 400 mL elution buffer to each of the input samples.CRITICAL: Carefully transfer the supernatant into a new 1.5 mL reaction tube without any beads.Use the 200 mL pipette tip to transfer the supernatant.f.Prepare fresh Reversion Mix Buffer and add 42 mL to each sample.g.Mix well by vortexing.h.Incubate for 16 h at 65 C. Note: Following 16 h incubation, you may store the samples at 4 C and purify the DNA up to two days later.10.Purify the DNA using the QIAGEN PCR Purification Kit.a. Mix each sample with 2.2 mL of Binding Buffer PB (in a 5 mL reaction tube) to obtain a clear suspension.b.Apply the sample to the provided QIAquick Spin Column.c. Centrifuge at 17,000 3 g for 1 min at 20 C-22 C and discard the flow through.d.Add 1 mL of PE Buffer to the wash.e. Centrifuge at 17,000 3 g for 1 min at 20 C-22 C. f.Transfer the filter to a new reaction tube.g.Add 30 mL of elution buffer per sample and incubate for 2 min at 20 C-22 C. h.Centrifuge at high speed for 2 min at 20 C-22 C.
Protocole.For transfection, dilute 40 mL Lipofectamine 2000 DNA Transfection Reagent in 750 mL serumfree Opti-MEM in a 2 mL reaction tube A. f.In a 2 mL reaction tube B add 750 mL Opti-MEM and 16 mg DNA (8 mg pEF1a-FB-dCas9-puro + 8 mg gRNA-MICA-T1 vectors).g.Mix both tubes by vortexing.h.Incubate the tubes for 5 min at 20 C-22 C. i. Add the content of tube A to tube B and mix by pipetting.j.Incubate for 20 min at 20 C-22 C. k.Add the transfection mixture dropwise to the 15 cm cell culture dish containing HEK293 cells in a circular motion.l.Incubate the dish at 37 C with 5% CO 2 for approximately 17 h.m.The next day, replace the transfection medium with DMEM medium containing 10% FBS and 1% penicillin/streptomycin. n.Evaluate transfection efficiency using pEGFP-C1 plasmid as a control and visualize by fluorescence microscopy.o.48 h post-transfection, subject cells to engineered DNA binding molecule-mediated chromatin immunoprecipitation (enChIP) assay.