CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

All animal experiments were conducted according to animal welfare regulations and have been approved by the responsible authorities (Regierungspräsidium Karlsruhe, approval numbers: G90/13, G176/13, G32/14, G48/14, and G133/14).

1. Generate pU6-sgRNA-Cbh-Cre Plasmids

1. Design the sgRNA to target a gene-of-interest according to the previously published protocol¹⁴.
   NOTE: In this experiment, two sgRNAs are designed to target the mouse Top2b gene and a non-targeted control sgRNA (Table 1). To knockout gene(s) using the CRISPR technology, sgRNAs need to be designed to target either the coding sequence of the 5' region or the essential functional domain of the protein. One convenient starting point is to test publicly available pre-designed sgRNA sequences, like the GeCKO v2 mouse knockout pool library¹⁵ from the Zhang lab. Alternatively, sgRNAs can be designed using public available software such as the following website: crispr.mit.edu.
2. Clone the sgRNAs into the pU6-sgRNA-Cbh-Cre vector.
   NOTE: The pU6-sgRNA-Cbh-Cre vector¹² used in this study is derived from the original pX330 plasmid¹⁶ by replacing the coding sequence of SpCas9 with the Cre recombinase.
   1. Synthesize two DNA oligos as follows. Sense: 5' – CACCGNNNNNNNNNNNNNNNNNNNN – 3'; Antisense: 3' – CNNNNNNNNNNNNNNNNNNNNCAAA – 5'.
      NOTE: The 20 N's shown above stand for the sgRNA sequence.
   2. Digest pU6-sgRNA-Cbh-Cre with BbsI for 30 min at 37 °C using the following reagents, load the digested sample to a 1% agarose gel, and purify them using a gel-extraction kit. Use 3 µg Plasmid; 3 µL BbsI; 1 µL FastAP; 5 µL 10X digestion buffer; add ddH₂O to bring the total volume to 50 µL.
   3. Phosphorylate and anneal each pair of sgRNA oligos using the following reagents (10 µL total volume): 1 µL Customized sense oligo (100 mM); 1 µL Customized antisense oligo (100 mM); 1 µL 10X T4 Ligation Buffer; 6.5 µL ddH₂O; 0.5 µL T4 PNK. Incubate in a thermo-cycler for 30 min at 37 °C, and 5 min at 95 °C, and then ramp down to 25 °C at 5 °C/min.
   4. Set up the following ligation reaction, and incubate for 10 min at room temperature (10 µL total volume): X µL BbsI digested plasmid (50 ng); 1 µL phosphorylated and annealed, oligo duplex (1:200 dilution); 5 µL 2X ligation buffer; X µL ddH₂O; 1 µL quick ligase.
   5. Add 2 µL of the ligation product into 50 µL of chemically competent DH5α Escherichia coli cells. After 30 min of incubation on ice, heat shock the sample for 90 s at 42 °C, and incubate for 2 min on ice. Add 100 µL of LB medium and plate it onto a LB plate containing 100 µg/mL ampicillin.
      NOTE: The integration of the sgRNA sequence into the pU6-sgRNA-Cbh-Cre plasmid is performed according to the cloning steps for the pX330 plasmid¹⁴.
   6. Inoculate two colonies, each in 2 mL of LB medium at 37 °C for a minimum of 6 h, and perform a mini-prep DNA isolation using a kit.
   7. Sanger sequence the mini-prep DNA using the hU6-Forward primer, and identify colonies with the correct insertion of sgRNAs by aligning with the sequence shown in step 1.2.1. The primer sequence is shown in Table 1. Plasmids used in this study were named pU6-sgTop2b-1-Cbh-Cre, pU6-sgTop2b-2-Cbh-Cre, and pU6-sgControl-Cbh-Cre.
   8. Inoculate the correctly-identified colonies and purify the endotoxin-free plasmid DNA for in utero electroporation using a kit, e.g., from Qiagen. Elute the DNA with endotoxin-free TE-Buffer diluted in ddH₂O at 1:10 (10% TE buffer). The DNA concentration of the plasmid needs to be higher than 2 mg/mL.
      NOTE: Do not use a regular maxi-prep kit for DNA preparation. Store plasmid-solutions at 4 °C for regular short-term use (up to 4 weeks), or aliquot an appropriate volume of about 25 µL and keep at -20 °C for long-term storage.

2. Test the Efficiency of the sgRNAs Using the EGxxFP Plasmid System

NOTE: The efficiency of the sgRNAs is normally tested by Surveyor or T7E1 (T7 endonuclease I) assays. In this protocol, an easy and efficient alternative approach is used. The key of this approach is to use the pCAG-EGxxFP plasmid which contains overlapping EGFP fragments separated by a DNA sequence containing the sgRNA targeting site¹⁷. Upon the expression of pCAG-EGxxFP together with the sgRNA and Cas9 in the transfected cells, the Cas9-mediated double strand break (DSB) in the target sequence is repaired by endogenous homology-dependent mechanisms, which reconstitutes the EGFP expression cassette.

1. Design primers to amplify the genomic region centered with the sgRNA targeting sequences. The PCR amplicon is approximately 500 bp. In this experiment, a DNA assembly was applied to clone a PCR-amplified fragment into the pCAG-EGxxFP plasmid.
   NOTE: Alternatively, appropriate restriction sites can be added to the 5' end of the PCR primers. Available restriction sites in the pCAG-EGxxFP vector are BamHI, NheI, PstI, SalI, EcoRI, and EcoRV.
2. Amplify the genomic DNA with designed primers using the form and PCR parameters below. Load the sample (19.7 µL H₂O; 0.3 µL of 10 ng/µL mouse genomic DNA; 2.5 µL 10 µM EGxxFP-Top2b-F; 2.5 µL 10 µM EGxxFP-Top2b-R; 25 µL 2X PCR master mix; 50 µL total volume) onto a 1% agarose gel and gel-purify the PCR fragments using a gel-extraction kit. Use the following reaction conditions:95 °C for 3 min; then 35x cycles of 98 °C for 20 s, 60 °C for 15 s, 72 °C for 20 s, and 72 °C for 1 min; 4 °C, indefinitely.
   NOTE: In this experiment, one region in Top2b exon 1 was amplified; find the primer sequences in Table 1.
3. Digest the pCAG-EGxxFP vector with restriction enzymes BamHI and EcoRI for 2 h at 37 °C, then load the sample onto a 1% agarose gel and gel-purify the vector backbone using a gel-extraction kit. Use the following reagents (50 µL total volume): X µL Plasmid (3 µg); 2 µL BamHI; 2 µL EcoRI; 5 µL 10X Buffer; X µL ddH₂O.
4. Set up the DNA assembly reaction¹⁸ according to the manufacturer's instructions, and transform into DH5α competent E. coli.
5. Inoculate two colonies, each in 2 mL of LB medium at 37 °C for a minimum of 6 h, perform a mini-prep DNA isolation using a kit, e.g., from Qiagen, and identify colonies with the correct insertion (hereafter referred to as pCAG-EG-Top2b-FP) using Sanger sequencing with the EGxxFP-Top2b-F primer.
6. Transfect HEK293T cells.
   1. Seed the HEK293T cells into a 24-well plate 24 h before transfection.
      NOTE: Cells were cultured in a 37 °C cell incubator with 5% CO_2, in DMEM with glutamine supplement containing 10% FBS and 1% Penicillin/Streptomycin. Ensure that the cells are 60% confluent on the day of transfection. In this experiment, we used self-made HEK293T cells stably expressing SpCas9 to test pU6-sgRNA-Cbh-Cre. However, wildtype HEK293T cells can be used if an SpCas9 expression vector is provided.
   2. Transfect the HEK293T cells using the polyethylenimine (PEI) reagent. Transfect the cells with 120 ng/well of pCAG-EG-Top2b-FP plasmid¹² and 120 ng/well of pU6-sgTop2b-1-Cbh-Cre or pU6-sgTop2b-2-Cbh-Cre in a 24-well plate. Mix the plasmid DNAs and the 1.8 µL of PEI reagent in 80 µL of unsupplemented DMEM medium with glutamine supplement and incubate for 15 min at room temperature after vortexing.
   3. 6 h after the transfection, remove the medium and replace with fresh medium.
   4. Monitor the presence of live GFP⁺ cells 48 h after transfection using a fluorescence microscope at 20x magnification.
      NOTE: The number of GFP⁺ cells indicates the targeting efficiency of the sgRNA. Results of effective and non-effective sgRNAs (sgTop2b-1 and sgTop2b-2, respectively) are shown in Figure 2A.

3. Perform In Utero Electroporation

1. Breed 6 to 8-week-old mice and prepare the tools for in utero electroporation. Cross male homozygous Rosa26-CAG-LSL-Cas9-P2A-EGFP mice with female CD-1 mice. Time the pregnancy of the CD-1 mice from the day the vaginal plug is detected (E0.5). Embryos are electroporated at day E13.5.
   1. Pull borosilicate glass capillaries (inside diameter: 0.6-0.8 mm) with a micropipette puller. Use the following settings: P = 500, Heat = 560, Pull = 150, Vel = 75, Time = 250. Label the capillary tip with black ink for better visualization during the injection. Trim the capillary taper under a stereomicroscope using a ruler and sharp needle tweezers and trim the tip at a length of about 6 mm.
   2. Maintain the unity of the capillaries to keep the experiment reproducible. Create a one-sided beveled edge during the trimming. The tip should be as sharp as possible for easy penetration of the uterine wall and to avoid tissue damage of the embryo.
      NOTE: Alternatively, use a micro grinder to adjust a 35° angle¹⁹. The capillaries can be stored at room temperature in a 10 cm Petri dish under pathogen-free conditions.
   3. Autoclave the surgical instruments.
   4. Mix the sgRNA-plasmids in equal parts with pT2K-IRES-Luc to a final concentration of at least 1 µg/µL for each plasmid and color the plasmid-solution with fast green (final concentration of 0.05%). Prepare 1% fast green stock solution with endotoxin-free distilled water and filter through a 0.22 µm filter to remove dye particles from the solution.
      NOTE: Co-transfection of pT2K IRES-Luc is optional, but allows for identification of viable electroporated animals after birth using bioluminescence imaging. Please see step 3.5. Prepare a sufficient amount of plasmid-mix for the total number of pregnant mice to be operated. Use about 15-20 µL mix per mouse (~ 12 embryos). Proceed immediately with the surgery.
2. Prepare mice for surgery.
   1. Anesthetize pregnant CD-1 mice with isoflurane. Induce anesthesia in a box with 3-4 vol-% isoflurane (oxygen flow rate: 1.5 L/min) and maintain it with 2 vol-% during surgery via nose cone.
      NOTE: The complete surgery should not take longer than 30 min. Reduce the number of injected and electroporated embryos, if necessary. Use sterile gloves for surgery.
   2. Place the mouse on its back on a heating pad and adjust anesthesia.
   3. Inject analgesic (Metamizol, 800 mg/kg body weight, subcutaneously (s.c.), 24 h depot).
      NOTE: You may use other authorized analgesics than Metamizol.
   4. Apply eye ointment to prevent eye-dryness during surgery.
   5. Fix limbs with tape and sterilize the abdomen with a disinfectant. Cover the mouse with gauze, leaving only the surgical area exposed. Spread 70% ethanol over surgical area and gauze.
   6. Make a skin incision of about 2 cm in length and widen the gap gently with straight surgical scissors. Locate the linea alba and make a slightly smaller second incision through the peritoneum using tissue scissors with blunt tip.
   7. Moisten the opened abdominal cavity with pre-warmed sterile 1x PBS.
   8. Carefully extract the uterine horns from the abdominal cavity using ring forceps. Pull gently by grabbing the uterine wall solely at the gap between the yolk sacs of two neighboring embryos.Avoid any pressure on the embryo while pulling it through the incision. Enlarge the incision if necessary and take extra care to position the uterine horns onto the abdomen while not compressing blood flow through the uterine artery.
      NOTE: In some cases, it is advised to extract one uterine horn at a time and replace it before proceeding with the second uterine horn.
3. Injection and electroporation of plasmid DNAs
   1. Aspirate approximately 15 µL of colored plasmid-solution with the prepared glass capillary. Avoid any air-bubbles during this process.
      NOTE: The plasmid-solution can clog the tip easily if its concentration is high. Test the capillary by releasing some DNA right before the injection.
   2. Gently hold the embryo with ring forceps and locate the neck area.
      NOTE: If the embryo is in a position difficult to inject, skip it and go for the next embryo. Increased repositioning of the embryo may increase chances of damage to them.
   3. Slowly pierce with the tip of the previously prepared glass capillary into the fourth ventricle by penetrating through the dorsal hindbrain and subsequently inject about 1 µL of the plasmid-mix. Confirm that the dyed DNA is not leaked from the brain and is visible as a diamond shaped structure.
      NOTE: As an option, use 2.5-fold magnifying glasses for better visualization of the fourth ventricle and surrounding blood vessels. Deliver electric pulses immediately after injection to prevent diffusion of the plasmid-solution to the other ventricles.
   4. Apply electric square pulses (5 pulses, 32 mV, 50 ms-on, 950 ms-off) with forceps-like platinum tweezers (see Table of Materials). Place the electrodes laterally with the negative pole covering the ear and the positive pole positioned at the cerebellar primordium over the uterine wall. Use 5 mm-diameter electrode plates for effective electroporation of E13.5 embryos.
      NOTE: Increase the survival rate of pups by manipulating less than 2/3^rd of the total number of embryos per dam. Avoid embryos too close to the cervix. If manipulated, they might cause complications during labor and jeopardize the entire litter. If electrodes are too close to the embryo's heart, this may cause cardiac arrest.
4. Post-electroporation and post-surgery care
   1. Carefully place uterine horns back into the abdominal cavity and fill with pre-warmed sterile 1x PBS. Let the uterine horns slide into a natural position.
   2. Close the peritoneum and skin separately with a simple continuous suture.
      NOTE: Take extra care not to pierce through the uterine wall when suturing the peritoneum.
   3. Shut-down the anesthesia and place the animal belly-down into a new cage.
   4. Monitor the animal during the waking phase and again 24 h after the surgery. Place the cage under a heating lamp, if the trembling doesn't improve within a 15 min time-frame and the animal doesn't start grooming.
   5. Confirm successful electroporation by luciferase imaging.
      1. Make sure that the operated dams drop embryos on time (at E19.5).
         NOTE: The birthdate may be delayed to the next day probably because of the operation.
      2. Anesthetize the electroporated pups (P5-P7) with 2.5% isoflurane and inject (intraperitoneal (i.p.)) with D-Luciferin (3 mg/kg body weight) 5 min prior to imaging.
      3. Detect luciferase signals in the electroporated pups using the bioluminescence imager.
         NOTE: A 1 min exposure time is sufficient to detect the luciferase signal. Radiance (p/s/cm²/sr) is approximately >1 x 10⁶.

4. Prepare Cryosections from the Electroporated Cerebella

1. Euthanize the electroporated P7 pups and dissect out the cerebellum.
   NOTE: The GFP signal can be observed using an epifluorescent stereoscope.
2. Fix the cerebella overnight in 5 mL per brain of 4% PFA in PBS at 4 °C.
3. Transfer the fixed cerebella overnight in 10 mL per brain of 30% sucrose in PBS at 4 °C.
   NOTE: The tissue should reach the bottom of a 15-mL tube after incubation in the sucrose solution.
4. After soaking up the remaining sucrose solution with a piece of cellulose filter paper, immerse the cerebellum in an optimal cutting temperature (OCT) compound in a disposable plastic mold and freeze it on dry ice. The cryogenic block can be stored at -80 °C until use.
   NOTE: The protocol can be paused here.
5. Cut the cryogenic block into sections with 10 µm in thickness using a cryostat and keep the sections at -80 °C until use.
   NOTE: One can confirm GFP expression in the sections, after washing out the OCT compound with PBS.

5. Immunostaining of the Cryosections

1. Dry the slides at room temperature for 30 min and wash twice for 10 min in PBS.
2. Circle the sections with a liquid blocker pen and incubate the sections in a blocking solution (10% normal donkey serum in PBS containing 0.1% Triton-X100) for 1 h at room temperature.
3. Add primary antibodies against the molecules of interest (e.g., GFP and topoisomerase II beta (Top2B) in this study) in the blocking solution and incubate overnight at 4 °C.
   NOTE: The names and concentrations of antibodies used are listed in the Table of Materials. In order to enhance GFP signals, an anti-GFP antibody can be optionally used together with the other antibodies.
4. Wash the slides with 0.1% Triton-containing PBST 3X for 10 min. Incubate the sections for 1 h at room temperature with a fluorophore-conjugated secondary antibody diluted in the blocking solution containing DAPI.
5. Wash the slides in PBS 3X for 10 min. Mount the slides and keep at 4 °C until imaging.
   NOTE: The protocol can be paused here.

6. Imaging and Analysis

1. Image the sections using a confocal microscope at 20x magnification.
2. Count the number of GFP positive cells losing the expression of the target protein. A representative image is shown in Figure 2B.
3. Check the molecular phenotype upon ablation of the target genes in GNPs and their progenies by immuostaining¹².