Gonad Microinjection: A Method of Compound Delivery Directly into the Germline of C. elegans

The following protocol is an excerpt from Farboud et al, Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms, J. Vis. Exp. (2018).

1. RNP Assembly

1. Design the experiment well in advance, acquiring all the RNA, DNA, and protein components ahead of time. As a first pass, try one of the positive controls listed in Table 1 and use the commercial reagents described in the Table of Materials to ensure a reliable experimental design and the integrity of the materials. For additional tips on planning a new genome-editing experiment, see papers on this topic.
   ​NOTE: Once assembled as described in the subsequent steps, RNPs prepared in advance may be stored at -80 °C.
   1. After choosing which gene to target, use one of the free online tools to design an optimal gRNA. Be sure to target an exon if hoping to generate a knockout.
      NOTE: These tools will help to identify a target site with an adjacent S. pyogenes PAM sequence, high-quality score, and low off-target score.
   2. Purify the S. pyogenes Cas9 protein through published methods or purchase it from a commercial vendor.
   3. Prepare a typical Cas9 buffer for the RNA dilution, RNP preparation, and protein storage, which contains 20 mM of HEPES pH 7.5, 150 mM of KCl, 10% glycerol, and 1 mM of TCEP. Always use nuclease-free water in buffers that will be used to resuspend or dilute RNA to prevent degradation.
   4. Produce the guide RNA (tracrRNA and crRNA or sgRNA) through an in vitro transcription using published methods, or purchase it from a nucleic acid synthesis company.
   5. If inserting a gene, synthesize or purchase a donor DNA template.
   6. Store the protein and RNA aliquots at -80 °C and thaw on ice immediately before use.
      ​NOTE: Each freeze-thaw slightly lowers the efficiency. Detailed, open-access protocols for Cas9 purification and the in vitro transcription of sgRNAs are available elsewhere.
2. RNP prep for C. elegans editing: Assemble the RNP complex by adding the following reagents in order to create a final volume of 20 µL (the final concentrations are noted in parentheses): Cas9 (2 µM), HEPES pH 7.5 (10 µM), KCl (115 µM), crRNA (12 µM), tracrRNA (40 µM), and the repair templates if needed (0.5 µM ssDNA or up to 350 ng/µL dsDNA).
   ​NOTE: The efficiency of a Cas9-mediated DSB-templated repair is proportional to the concentration of the dsDNA repair construct; thus, the higher the concentration of the repair template, the more efficient the templated repair. However, an injection of mixes containing greater than 350 ng/µL of dsDNA has been shown to reduce the viability of the injected worms. Thus, it is best to use up to, but no more than 350 ng/µL of dsDNA in the mix to maximize the repair efficiency while minimizing its lethality.
   1. Add multiple crRNAs to target multiple loci simultaneously, as needed for the co-CRISPR/co-conversion screening approach. When adding more than one crRNA, add each sequentially to the master mix.
      ​NOTE: The amount of each crRNA does not need to be the same, and even doubling the total concentration of crRNAs in the master mix without changing the concentration of Cas9 does not appear to interfere with the frequency of mutagenesis at a specific locus. Examples are described in detail in Paix et al.
   2. Mix by pipetting and spin the RNP solution at 16,000 x g for 5 s to ensure that the solution is collected at the bottom of the tube.
   3. Incubate the solution at 37 °C for 15 m.
   4. Centrifuge the sample at 16,000 x g for 1 min to pellet any particulates that could clog the thin-bored microinjection needle. Use the supernatant in the subsequent steps.
3. C. elegans Preparation

1. 1 day prior to microinjection: Prepare the agarose pads for the microinjection.

1. Make a 3% (w/v) agarose solution in water by adding agarose to water and bringing the solution to a boil on a hot plate or in a microwave.
2. Arrange 24 mm x 50 mm x 1.5 mm cover glass slides on a table and use a glass Pasteur pipette to place a small (~15 µL) drop of agarose solution onto the slide. Quickly flatten the agarose drop by placing another coverslip on top. Allow the agarose to solidify and then remove one of the coverslips.
3. Leave the agarose-coated coverslip face-up on a tabletop overnight to dry. After 24 h, store the agarose pads in a clean, dry container.
   NOTE: These can be used indefinitely.

2. Pull the microinjection needles: using borosilicate glass capillaries with filaments (outer diameter 1.0 mm and inner diameter 0.58 mm), pull the needles based on Mello and Fire⁵⁷ and other resources. The needles can be used immediately or can be stored in a clean, dry container, braced by clay supports.

3. For the maintenance of the worms, prepare a Nematode Growth Media (NGM) agar poured into Petri plates and spotted with OP50 bacteria (for protocols on standard C. elegans maintenance and recipes for growth media, see Stiernagle).

4. Stage the worms for microinjection: 12-24 h prior to the microinjection, pick L4-staged hermaphrodites to a new NG-agar plate with OP50 bacteria and incubate them overnight at 20 °C. For each Cas9 target/injection mix, pick ~30 worms to the plate.

4. Day of microinjection: Load the pulled microinjection needle with the RNP solution supernatant prepared in step 1.2.
   1. Pipette the supernatant from step 1.2.4 into a pulled capillary pipette and backfill the solution from the capillary pipette into the prepared microinjection needle (generally loading less than 0.1 µL).
5. Mount the loaded needle onto the microinjection apparatus attached to a micromanipulator. Set the injection apparatus pressure to 250 kPa and the balance pressure to 25 kPa.
6. Break back the loaded needle tip to generate a sharp needle edge. Place a 15 mm x 15 mm x 1.5 mm square coverslip on the top of a 24 mm x 50 mm x 1.5 mm coverslip.
   1. Overlay one edge of the square coverslip with halocarbon oil 700.
   2. Position the needle in the oil, at the edge of the 15 mm square coverslip.
   3. Using a hand to guide the microscope stage and coverslip, brush the slide up and along the edge of the needle while depressing the injection pedal/button. Break the needle tip back, increasing the flow of the liquid out of the needle. Achieve an optimal flow rate by making the injection mix flow up along the edge of the needle, forming ~1 bubble/s.
7. Confirm that the L4 worms picked 12-24 h prior to microinjection are developmentally staged young adults on the day of injection. Pick the young adult worms to an NG-agar plate that lacks OP50 bacteria and allow them to crawl around for 5 min. This reduces the quantity of bacteria transferred to the injection pad, minimizing needle clogs.
8. Place an agarose injection pad/coverslip onto a dissection scope. Using a worm pick, lay a small track of halocarbon oil along one edge of the pad.
9. Using the worm pick coated in oil, lift several worms off the NG-agar plate and into the track of oil. With a fine hair attached to a pipette, such as an eyelash or cat whisker, position the worms in parallel, gently pushing the worms into the agarose pad. Until comfortable with the microinjection procedure, only mount and inject one worm at a time.
   ​NOTE: The dry agarose will wick the moisture from the worms, causing them to adhere to the pad. Consequently, one must work quickly as the worms can desiccate.
   1. Once in position and attached to the pad, overlay the worms with another few drops of halocarbon oil (~20 µL) from the tip of the worm pick.
10. C. elegans Gonad Microinjection with RNPs and Post-injection Care
    The microinjection protocol is adapted from Mello and Fire and described in detail elsewhere.
    1. Place the coverslip with the mounted worms onto the injection microscope. Under a low magnification (5X objective, 10X ocular), position the worms perpendicular to the injection needle.
       1. Switch to a high magnification (40X objective, 10X ocular), reposition the needle adjacent to the gonad arm corresponding to the region near the nuclei in mid- to late-pachytene.
       2. Using the micromanipulator, move the needle against the worm, depressing the cuticle slightly. Then, with one hand, tap the side of the microscope stage to jolt the needle through the cuticle. Depress the injection pedal/button and slowly fill the gonad arm with the injection mix and remove the needle.
       3. Repeat this step with the other gonad arm.
    2. Once the worms are injected, remove the coverslip/agarose pad and place it under a dissecting microscope.
       1. Using a pulled capillary pipette, displace the oil from the worms by pipetting an M9 buffer over them. Perform this treatment to release the worms from the agar.
       2. After 10 min, when the worms are thrashing around in the buffer, move them to an NG-agar plate with OP50 bacteria using the pulled capillary pipette. Place the plate at 20 °C for 2-3 h until the worms have recovered and are moving around.
    3. Once recovered, individually transfer the worms to NG-agar plates with OP50 and transfer the plates to a 25 °C incubator.
    4. Allow the P₀-injected worms to grow and lay progeny for 3 days. Screen the F₁ offspring.
       1. If using co-CRISPR or co-conversion, then select the candidate worms for screening based on whether they have the mutant phenotype of the reference gene. Individually transfer these marked worms to new NG-agar plates with OP50 and allow them to lay F₂ progeny at 20 °C.
          NOTE: The phenotype used for a co-CRISPR screening or selection should provide an early estimate for the success of Cas9 editing.
       2. If the co-CRISPR phenotype is not present, microinject a positive control plasmid to assist in improving the microinjection efficiency.
          NOTE: For instance, including a plasmid in the injection mix that encodes mCherry-tagged MYO-2 will help assess the injection efficiency. Worms successfully injected with pCFJ90 will have some offspring with fluorescent pharynxes.
    5. Examine the F₁ worms for the presence of the desired edits. Pick the F₁ mother to an individual well of a 96-well plate, lyse her, and examine her DNA by either insert-specific PCR amplification, DNA sequence analysis, or surveyor nuclease assay (CEL-1).
       NOTE: These assays can be performed when using a co-CRISPR/co-conversion or other screening or selection regimes.