Brown Adipose Tissue (BAT), specializes in energy dissipation, and also functions as an endocrine organ secreting bioactive molecules. Generating BAT-specific knockout mice is a predominant approach for elucidating gene contributions to BAT-mediated energy regulation. This protocol delineates a strategy integrating Cre-LoxP, CRISPR-Cas9, and Adeno-Associated Virus-single guide RNA (AAV-sgRNA) systems for rapid and efficient gene knockout within BAT. Precise surgical procedures are paramount to prevent neural and vascular damage. Proficiency in BAT anatomy and surgical skills is pivotal to minimizing tissue injury. The protocol emphasizes key technical steps encompassing sgRNA design, AAV-sgRNA particle preparation, and direct AAV microinjection into BAT, facilitating widespread application in BAT gene function studies.

Brown Adipose Tissue (BAT) is an endocrine organ that secretes bioactive chemicals and specializes in energy dissipation. Creating BAT-specific knockout mice is a common method used to identify the genes involved in BAT-mediated energy control. This technique describes a method for quick and effective gene knockdown in BAT using the Cre-LoxP, CRISPR-Cas9, and Adeno-Associated Virus-single guide RNA (AAV-sgRNA) systems. Accurate surgical techniques are essential to avert brain and blood vessel injury. To minimize tissue damage, surgical expertise and BAT anatomical proficiency are essential. In order to enable broad use in BAT gene function research, the technique places special emphasis on crucial technical processes pertaining to sgRNA design, AAV-sgRNA particle manufacturing, and direct AAV microinjection into BAT.

Experimental Materials and Recommended Services

Procedure

1. Screening Effective sgRNAs in Cultured Cells

a. CRISPR-Cas9 sgRNA Design

• To design sgRNAs, use online resources like the Broad sgRNA design tool e Biogene (Using CRISPR-ERA Webserver for sgRNA Design Protocol - Creative Biogene) and other tools that are accessible. Furthermore, Creative Biogene offers efficiency-validated sgRNA design services.
• To produce prospective sgRNA sequences, enter gene names or DNA target sequences into the designated box and choose NGG as the protospacer adjacent motif (PAM) for SpCas9.
• Give sgRNAs with minimal off-target activity and high anticipated on-target efficiency priority. For example, it is advised to use sgRNAs whose specificity score on the CRISPR output is at least 50. Choose the sgRNAs with high efficiency ratings from those that match this requirement.

b. Construction of the CRISPR-Cas9 sgRNA Plasmid

• Using a DNA synthesis platform, create sgRNA oligo pairs that encode 20 nucleotides (nt) targeted sequences with overhangs (both 5' and 3') from the BsmBI restriction site.
• Ligase-annealed oligo pairs using lentiCRISPR v2 vector that is BsmBI-linearized.
• Use the U6 forward primer to validate sgRNA insertions by Sanger DNA sequencing after transforming the ligation product into the Stbl3 E. coli strain.

c. Producing Lentiviral Particles

• 24 hours before to transfection, seed about 7 × 105 HEK-293 cells in a 6 cm tissue culture plate with 4 mL of full growth media.
• Combine 250 µL of serum-free media with 15 µL of LT1 transfection reagent, then let it sit at room temperature for five minutes.
• For every sgRNA, prepare a transfection cocktail in 250 µL of serum-free media. After mixing the transfection reagent with the plasmid cocktail, let it sit at room temperature for 20 to 30 minutes.
• For HEK-293 cells, replace the entire growth media with 3.5 mL of new growth medium. Drop by drop, add the DNA transfection reagent combination to the cells growing in the new growth media.
• Replace the medium to eliminate the transfection reagent and add 4 milliliters of new growth media after transfection for a duration of 12 to 15 hours.
• Following a 24-hour incubation period, extract the lentiviral particle-containing cell culture media and pass it through a 0.45 µm filter to exclude any HEK-293 cells. For short-term usage, keep the viruses at 4°C; for long-term storage, freeze them at -80°C.

d. Infection of Brown Preadipocytes and Determination of sgRNA-Mediated Knockdown

• Place one million mouse immortalized brown preadipocytes in a 6-well plate with 1 mL of new media containing 8 µg/mL polybrene each well.
• Apply a 1 mL lentiviral particle solution to infect cells. Keep a well of healthy cells as a control when choosing antibiotics.
• A new medium should be used 24 hours following infection. To identify the infected cells, add the appropriate antibiotics (puromycin, for example, at a final concentration of 1 µg/mL). Until every other day, switch to a new medium containing antibiotics and wait for all the uninfected control cells to die.
• Using western blot analysis, extract protein from virus-infected cells and assess the effectiveness of sgRNA knockdown. Test at different times to see if there is a loss of protein signal as a result of different turnover rates (for example, from day 6 to day 12 following viral infection).

Figure 1: Schematic for the lentiviral and the AAV plasmid expressing Cas9 and sgRNA. (Tsuji, T., et al., 2023)

2. Construction of the AAV-sgRNA Plasmid

a. The pAAV-U6-BbsI-gRNA-CB-EmGFP backbone should be linearized using BbsI, which produces overhangs that are similar to those of manufactured sgRNA oligos.

b. Verify sgRNA insertions by ligand-annealing oligo pairs with linearized pAAV vector.

3. AAV Packaging

a. Pack AAV vectors into AAV serotype 8 that were created in part 2. Make high-titer AAV by an earlier technique or ask commercial suppliers or viral cores for AAV packaging services. Put 1 x 1013 genome copies/mL in the viral titer.

4. Preparation of Ucp1 Cre/Cas9 Mice

a. Acquire a mouse strain bearing the Ucp1 promoter and expressing Cre recombinase. Obtain homozygous Rosa26-floxed STOP-Cas9 knock-in mice, in which the production of Cas9 is reliant on the Cre recombinase enzyme.

b. Combine these strains to create mice that have Ucp1-Cre/Cas9. House mice in a controlled habitat with access to food and water, as well as regulated light and dark cycles, temperature, and humidity.

b. Give Ucp1-Cre/Cas9 mice AAVs containing either control or gene-targeting sgRNA.

5. Surgery for AAV Injection into BAT in Mice

a. Constantly inhale 2.5% isoflurane into mice's respiration to induce and maintain anesthesia.

b. Use analgesics (subcutaneous injection of Banamine, 2.5 mg/kg BW) to reduce discomfort following surgery.

c. Shave and sterilize the operative location to get it ready for surgery. Cut skin to reveal BAT.

d. After successfully injecting 40 µL of AAV into each BAT lobe, validate the injection.

e. Keep an eye out for bleeding and provide hemostasis as needed.

f. Seal off the area where the incision was made, and make sure the animals heal on a heating pad with bedding, drink, and food available.

b. After the mice have had a week to recuperate, keep an eye out for any indications of suffering or disease.

h. Verify the effectiveness of gene deletion in mouse BAT dissections using western blot analysis.

Figure 2. AAV injection into the bilateral BAT lobes of mice via sequential surgery. The following procedures were performed on an anesthetized mouse: (A–B) skin incision; (C–D) gentle skin removal; (E–F) cutting; (G) gentle removal of fat tissue; (H–I) injection of AAV into BAT lobes using a Hamilton syringe; (J) suturing between fat tissues and muscle; and (K–L) suturing between skin flaps. Note: Sultzer's vein verifies the placement of the BAT in (H) and (I). These panels have totally exposed BAT. The injection location in (I) and the adipose tissue-muscle boundary in (E) are shown by yellow lines, respectively. (Tsuji, T., et al., 2023)

This protocol underscores several critical steps for success, including sgRNA design, AAV packaging and concentration, generation of BAT-specific Cas9 mice, and surgical procedures. Superior surgical proficiency is necessary, especially when it comes to reducing tissue damage during AAV injection and BAT exposure. A variety of environmental and genetic variables, including nutrition, exercise regimen, and housing temperature, can have a complex impact on an animal's BAT composition and amount of surrounding white fat.