Mar 26, 2026
In Vivo Smooth Muscle Cell-Specific CRISPRi Perturb-seq - Baseline Condition (4 weeks no diet, no ApoE)
- João Pinho Monteiro1,
- Daniel Yuhang Li1
- 1Stanford University School of Medicine
Protocol Citation: João Pinho Monteiro, Daniel Yuhang Li 2026. In Vivo Smooth Muscle Cell-Specific CRISPRi Perturb-seq - Baseline Condition (4 weeks no diet, no ApoE). protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3mneel25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 26, 2026
Last Modified: March 26, 2026
Protocol Integer ID: 313920
Keywords: cell transcriptional profiling during atherosclerosis, vivo smooth muscle cell, functional genomics in vivo, adult smcs via tamoxifen administration, dcas9krab mice, targeting gene, targeted gene repression, smooth muscle cell, sgrna, cell rna, traced functional genomic, tamoxifen induction, specific crispri perturb, cell suspension, tamoxifen administration, single systemic aav injection via retro, transcriptional profiling, single systemic aav injection, gene regulatory perturbation, crispr, vivo, week after tamoxifen induction, aortic root
Funders Acknowledgements:
IGVF
Grant ID: HG011972
Abstract
This protocol describes an in vivo smooth muscle cell (SMC)-specificPerturb-seq workflow enabling targeted gene repression and single-cell transcriptional profiling during atherosclerosis. Using Myh11CreERT2; ROSAtdT/+; dCas9KRAB mice, dCas9-KRAB expression is induced in adult SMCs via tamoxifen administration. One week after tamoxifen induction, mice receive a single systemic AAV injection via retro-orbital delivery under isoflurane anesthesia to introduce sgRNAs targeting genes of interest or non-targeting controls. At defined endpoints, aortic roots are harvested and enzymatically dissociated to generate single-cell suspensions. Dual-positive tdTomato⁺/GFP-reporter⁺ cells are isolated by FACS and processed using the 10x Genomics Chromium platform for single-cell RNA sequencing and CRISPR guide capture. This integrated workflow enables high-resolution, lineage-traced functional genomics in vivo, allowing assessment of SMC state transitions and gene regulatory perturbations.
Troubleshooting
In Vivo Smooth Muscle Cell-Specific CRISPRi Perturb-seq - Baseline Condition (4 weeks no diet, no ApoE)
Abstract (JPM Edited 2/25/26)
This protocol describes an in vivo smooth muscle cell (SMC)-specificPerturb-seq workflow enabling targeted gene repression and single-cell transcriptional profiling during atherosclerosis. Using Myh11CreERT2; ROSAtdT/+; dCas9KRAB mice, dCas9-KRAB expression is induced in adult SMCs via tamoxifen administration. One week after tamoxifen induction, mice receive a single systemic AAV injection via retro-orbital delivery under isoflurane anesthesia to introduce sgRNAs targeting genes of interest or non-targeting controls. At defined endpoints, aortic roots are harvested and enzymatically dissociated to generate single-cell suspensions. Dual-positive tdTomato⁺/GFP-reporter⁺ cells are isolated by FACS and processed using the 10x Genomics Chromium platform for single-cell RNA sequencing and CRISPR guide capture. This integrated workflow enables high-resolution, lineage-traced functional genomics in vivo, allowing assessment of SMC state transitions and gene regulatory perturbations.
We have obtained several lines of genetically modified mice from other laboratories, and combined alleles to undertake experiments outlined in here. To lineage trace vascular smooth muscle cells (SMC), we employed a SMC-specific mouse Cre allele (Myh11CreERT2; TgMyh11-CreERT2; 019079; JAX) combined with a Cre-responsive reporter gene (tandem dimer Tomato, tdT) inserted at the ROSA26 locus (ROSAtdT/+; B6.Cg-Gt(ROSA)26Sortm14(CAGtdTomato)Hze/J; 007914; JAX).
Adding a conditional dCas9-KRAB knock-in (dCas9KRAB; B6.129S6-Gt(ROSA)26Sortm2(CAG-cas9*/ZNF10*)Gers/J; 033066; JAX) mutation with these alleles has created the experimental mice, Myh11CreERT2, dCas9KRAB, ROSAtdT/+ that are both lineage traced for the SMC lineage and have SMC specific expression of dCas9-KRAB. All mice are on C57Bl/6 background.
Detailed Description of dCas9-KRAB knock-in allele -dCas9-KRAB knock-in mice (also called R26-LSL-dCas9-KRAB) have a loxP-flanked STOP cassette (all inserted into the Gt(ROSA)26Sor locus) preventing widespread expression of the RNA-guided DNA-binding fusion protein dCas9-KRAB - a human codon-optimized, nuclease-deficient, catalytically dead Cas9 protein (dCas9) fused to a Krüppel-associated box domain (KRAB). Following Cre recombinase exposure and introduction of a single guide RNA (sgRNA) that directs dCas9-KRAB binding to a specific DNA sequence/locus, the KRAB domain subsequently induces silencing of the target locus via heterochromatin formation and/or DNA methylation.
Published work from the Gersbach Lab (PMID: 34341582) reports that when using dCas9-KRAB in conjunction with Cre recombinase expression, the resulting mice all show the desired dCas9-KRAB expression levels, with appropriate target gene repression/methylation as determined by the sgRNA used.
1) Cre induction to activate reporter gene and dCas9-KRAB in SMC
Reagents
- Tamoxifen (Sigma-Aldrich; CAS T5648-1G)
- Sunflower seed oil (Sigma-Aldrich; CAS S5007-259ML)
- 100% Ethanol
- Oral gavage needles (appropriate gauge/length for adult mice) + syringes
1.1 Tamoxifen solution preparation (20 mg/mL)
1. Prepare vehicle (oil/EtOH) by mixing 9/10 sunflower oil and 1/10 ethanol (e.g. 900 µL oil + 100 µL EtOH).
2. Weigh tamoxifen to make a 20 mg/mL final concentration (e.g add 1000 µL of the oil/EtOH vehicle to 20mg tamoxifen).
3. Allow mixture to dissolve overnight at 37°C until fully in solution.
4. After dissolved, store at 4°C for up to 1 week.
1.2 Induction regimen (dose and schedule)
- Initiate Cre-Lox recombination at 7-8 weeks of age.
- Administer tamoxifen twice by oral gavage at ~0.2 mg/g body weight, with the two doses separated by 72–96 hours.
Working example:
Adult mouse ~30 g → required dose = 0.2 mg/g × 30 g = 6 mg
Using 20 mg/mL stock → volume = 6 mg ÷ 20 mg/mL = 0.3 mL (300µL)
2) AAV injection
Timing and dosage:
- Perform retro-orbital AAV-PR injection at 8 weeks of age
- Use a single dose of AAV-PR at 1×10¹² vg/mL, delivered at 100 µL per mouse.
Reagents:
- AAV (stock titer as provided)
- We use the AAV-PR for its smooth muscle tropism
- Sterile DPBS
- Isoflurane
- 0.5% ophthalmic Proparacaine
Equipment:
- U-100 BD Ultra-Fine‱ Short Insulin Syringes, 31G (BD Medical, #BD328438)
- Isoflurane vaporizer + induction chamber + nose cone
- Heat pad
2.1) Prepare virus:
Thaw AAV on ice. If you don’t use a full vial, aliquot and immediately refreeze on dry ice and store at -80C.
Prepare virus dilution in sterile DPBS to reach the required dose of 1×10¹² vg/mL in a final volume of 100 µL per mouse.
Load 100 µL into a 31G insulin syringe. Minimize bubbles.
2.2) Retro-orbital injection procedure:
Place one mouse into an induction chamber.
Start isoflurane at 3-4% with a flow rate of 0.8–1.0 L/min.
When the breathing rate slows to ~1 breath/second, proceed.
Maintain anesthesia (isoflurane at 1-2%) with a nose cone as needed while you perform the injection.
Place mouse on its side and press back on the upper and lower eyelids, slightly protruding the globe. To provide post-procedural analgesia, a drop of 0.5% proparacaine HCl ophthalmic solution can be placed in the eye that will receive the injection. After a few seconds, gently dab the eye with a sterile gauze to absorb any excess solution.
Using a 1 cc syringe or smaller and a 27-30 g, ½" needle, insert the needle at a 30-45° angle to the eye, bevel down, lateral to the medial canthus, through the conjunctival membrane.
Advance the needle until the bony orbit is reached but not penetrated. The needle is slightly retracted and the substance is injected slowly and smoothly mid-sinus (total 100 µL).
Remove the needle slowly and smoothly to prevent injury to the eye close the eyelids, and apply mild pressure to the injection site with a gauze sponge.
Allow the mouse to recover in a paper-lined cage on top of a heating pad; mouse is monitored every 15 minutes until ambulatory.
3) Tissue Collection and Liberase Based Cell Digestion ProtocolUntitled section
Endpoint :
- Sacrifice at 5 weeks following the schedule above.
Reagents:
- Liberase TM medium Thermolysin (Roche #05401127001)
- Reconstituted in HBSS to 26 units/mL.
- After reconstitution, divide into single-use aliquots, as performance degrades significantly with
freeze-thaw.
- Elastase (Worthington #LS002279), stock solution has variable concentration
- HBSS, calcium, magnesium, no phenol red (Gibco # 14025092)
- PBS
Equipment:
- Fine dissection scissors (e.g. FST 14058-09)
- 5mL Round Bottom Polystyrene FACS Tubes w/ 35um Strainer Cap (Falcon # 352235).
- Pre-cooled centrifuge at 4°C w/ swing bucket
3.1) Procedure:
1) Prepare the Enzyme Solution (on ice):
Final (working) concentration of Liberase is 2 units/mL
Final (working) concentration of Elastase is 2 units/mL
For 1mL of enzyme solution:
Liberase TM (26 units/mL) 77 uL
Elastase (125 units/mL) 16 uL
HBSS 907 uL
Total 1 mL
2) Place enzyme mixture on ice while aorta is being harvested
3) Terminal anesthesia and perfusion:
Anesthetize the mouse with isoflurane (3–4%, 0.8–1.0 L/min) until surgical depth is confirmed.
Perform a midline skin incision over the abdomen and thorax, open the abdominal cavity, puncture the diaphragm, and complete thoracotomy. Incise the right atrium to allow exsanguination. Insert a 25–27G needle into the left ventricle or ascending aorta and perfuse with ~10 mL sterile PBS until tissues blanch and effluent runs clear. Proceed immediately to vascular tissue collection.
4) Harvest aortic root and open longitudinally. Wash with PBS x3 in 12-well dish. Keep on ice while collecting from multiple mice.
5) Move root to 12-well dish with 1mL of enzyme mixture.
6) Incubate in 37C incubator for ~30-45 minutes (adjust time based on age and extent of calcification e.g. if ApoE-null with 16wk high fat diet, consider incubation for 45 minutes). At the end of the incubation, mince thoroughly with scissors while on ice for a few minutes (no more than 5min) until tissue is mostly dissolved.
7) Pipet up and down ~20-30 times to thoroughly disrupt tissue. Confirm full digestion under the microscope (single cells w/o clumps). If the tissue is not fully digested after pipetting incubate at 37C for an additional 10-15 minutes (Not recommended prolonged incubation as it may lead to increased cell lysis).
8) Transfer cell suspension into 1.5mL Eppendorf and centrifuge at 500xg for 5 minutes.
9) Carefully remove supernatant (confirm that the cell pellet is still present).
10) Resuspend the cells in 300uL HBSS (adjust volume as needed).
11) Immediately prior to FACS and/or staining, pass cells through a 35um strainer.
Note: Coating collection tubes in 10% BSA solution may improve cell survival.
12) Sort dual positive tdT+ (lineage-traced SMC-derived) and GFP+ (AAV transduced) cell population by FACS.
4) 10x Genomics library construction + sequencing
- Chromium GEM-X Single Cell 3’ Kit v4 (PN-1000691)
- Chromium GEM-X Single Cell 3’ Gel Bead v4 (PN-2001128)
- Chromium GEM-X Single Cell 3’ Chip Kit v4 (PN-1000690)
- Dual Index Kit TT Set A (PN-1000215)
- Dual Index Kit NT Set A (PN-1000242)
- 3’ Feature Barcode Kit (PN-1000262)
Target empirical performance/scale described:
· routinely >8,000 cells per run
· pool dual positive tdT+/GFP cell from 3–5 mice per capture to ensure
adequate cells
Procedure :
Followed standard protocol from 10x Genomics.
Chromium GEM-X Single Cell 3' v4 Gene Expression User Guide, CG000731
For Feature Barcodes
Chromium Single Cell 3' Reagent Kits User Guide (v3.1 Chemistry Dual Index) with Feature Barcoding technology for CRISPR Screening and Cell Multiplexing, User Guide, CG000389. Follow Step 2.3C followed by Step 4 for guide library construction.