A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Mutations in human superoxide dismutase 1 (SOD1) account for ~10 – 20% of familial amyotrophic lateral sclerosis, a devastating neurodegenerative disease that invariably leads to paralysis and death¹⁷. Human SOD-1 is an evolutionarily conserved protein sharing 55% identity and 70% similarity with the C. elegans SOD-1 protein (Figure 1B). To demonstrate the simplicity, feasibility, and efficiency of the CRISPR-Cas9 RNP-based approach, we targeted the C. elegans sod-1 gene to introduce the disease-associated human G93A variant in the worm genome (Figure 1A).

To engineer the G93A mutation in the C. elegans genome, an sgRNA was chosen whose PAM recognition site sits 3 bp upstream of the G93 codon (Figure 1C). We designed an ssODN HDR repair template containing: 1) nucleotide changes to convert the G93 codon to A93 (GGA GCA), 2) a silent change to the NGG PAM sequence (CGG CAG) to prevent sgRNA-Cas9-mediated cleavage of the HDR template, 3) a silent mutation (CT) that introduces a unique HindIII restriction enzyme site for genotyping, and 4) 50 bp 5' and 3' homology arms (Figure 1C). A PCR primer set (F1-R1) was designed to produce a single 592 bp PCR product in N2 controls (Figure 1A). An injection mix containing the sgRNA, Cas9, ssODN and a fluorescent marker plasmid (pCFJ90) were mixed together, incubated for 10 min at room temperature, and loaded into a microinjection pipette.

Figure 1D depicts the workflow after the injection mix is loaded into an injection micropipette. On Day 0, 10 – 15 P₀ animals were injected in one or both gonad arms using the standard microinjection technique^18,19. Injected animals recovered for 1 – 2 h and were then individually plated on OP50-seeded NGM plates. After 2 – 3 days, successful P₀ injections were identified by those having mCherry(+) F₁ progeny. The top three P₀ plates (i.e. those having the greatest number of mCherry(+) F₁s) were chosen for subsequent analysis. From each of these three plates (P₀-8, P₀-10, P₀-12), 8 mCherry(+) F₁s were singled to individual OP50-seeded 35 mm NGM plates for a total of 24 mCherry(+) F₁s. After 2 - 3 days of egg-laying (Day 4 – 5), the individual F₁s were transferred into PCR tubes containing worm lysis buffer and frozen for 1 h at -80 °C. Subsequently, the F₁s were lysed to liberate genomic DNA, and subjected to PCR. The PCR products were then purified and digested with HindIII for 1 h, and run on a 1.5% agarose gel to identify potential edited animals. In correctly edited animals, HindIII digestion cuts the full-length PCR product (592 bp) into two bands of 370 bp and 222 bp. This genotyping strategy unambiguously differentiates between wild-type (592 bp), heterozygote (592, 370, 222 bp) and homozygote (370, 222 bp) animals. Figure 2A illustrates the genotyping results for the 24 mCherry(+) animals.

To demonstrate that the fluorescent mCherry marker enriches for genome modification, we also picked 8 mCherry(-) animals from each of the three P₀ plates. Of the 24 mCherry(+) F₁s screened (red bar), 10 contained mono-allelic or bi-allelic modification (42%), 10 were wild type (42%), 3 were potential indels (13%), and there was 1 PCR failure (Figure 2A). In contrast, of the 24 mCherry(-) F₁s screened, only 1 animal was correctly modified (4%); whereas, 23 were wild type (96%) (Figure 2A). Figure 2B shows the chromatogram from the full-length gel extracted PCR product (F₁8-6), demonstrating that the precise nucleotide changes designed in the ssODN HDR template were faithfully incorporated into the genome of this homozygous F₁ animal. Notably, F₁ animals 10-7, 10-8, and 12-3 exhibited unexpected PCR product sizes and restriction digest banding patterns, suggesting these animals may carry complex indel mutations (Figure 2A). Thus, our method is not only capable of generating desired genome modification(s) with ease, efficiency, and fidelity, but also permits the identification and recovery of unique indel alleles that may shed important and unexpected insight into gene function.

Figure 1. CRISPR-Cas9 engineering of the C. eleganssod-1 locus. (A) Schematic illustration depicting the exon-intron structure of the sod-1 locus in C. elegans. The red bar denotes the location of G93 in exon 3. The primer pair set is noted by the red arrows (F1-R1). (B) Sequence alignment of human and C. elegans (worm) SOD-1 proteins. The targeted G93 residue is highlighted in red. (C) Top, a section of genomic DNA surrounding the G93 codon is shown as a reference, and the PAM (green) and sgRNA target (blue) sites are highlighted. Bottom, the single stranded oligonucleotide (ssODN) homology directed repair (HDR) template is shown containing: the codon edit changing G93 to A93, a silent change to the PAM motif to prevent cleavage by the sgRNA-Cas9 complex, a silent change to introduce a unique HindIII restriction enzyme site (yellow box), and flanking 5' and 3' 50 bp homology arms (black bars). All nucleotide changes designed in the ssODN are highlighted red. (D) Schematic illustration of the pipeline for generating and identifying CRISPR-Cas9 RNP-based point mutants. On day 0, inject 10 – 15 P₀ animals with injection mix. On day 2 – 3, identify successfully injected P₀ animals by those containing fluorescent F₁ progeny, and select three P₀ plates with the most fluorescent progeny. From each of the three P₀ plates, single 8 fluorescent F₁ progeny. On day 4 – 5, (a) step 1, individual F₁s are picked and placed into PCR tubes containing Lysis Buffer; (b) step 2, after freezing at -80 °C, PCR tubes containing F₁ worms are lysed to release genomic DNA, which is used as a PCR template. The PCR products are then cleaned and digested with the unique restriction enzyme; (c) step 3, enzyme digested products are resolved on an agarose gel where wild type (+/+), heterozygous (m/+), and homozygous (m/m) animals can be identified by restriction digest banding patterns. Please click here to view a larger version of this figure.

Figure 2. Genotyping data for sod-1 (G93A) mutants.(A)Gel image of F₁ animals that were individually lysed, PCRed and digested with HindIII. For each P₀ plate (P₀-8, P₀-10, P₀-12), 8 mCherry(+) and 8 mCherry(-) F₁ animals were analyzed. Both mono-allelic (blue numbers) and bi-allelic (red numbers) edited animals were recovered. Size shifted PCR products or unpredicted HindIII digested bands were also recovered that are indicative of indel mutants (yellow numbers). N2 controls are shown as a reference for PCR product sizing and enzyme specificity. (B) Top, codon spacing and amino acid translations are shown above for the wild type (WT) and below for G93A modified strands. The desired edit is highlighted by a red box, and the silent changes that create an in-frame HindIII restriction site are highlighted by a yellow box. All designed nucleotide changes are shown in bold. Bottom, representative chromatogram from homozygous F₁ animal 8-6. Please click here to view a larger version of this figure.

Table 1. CRISPR-Cas9 RNP Injection Mix. All reagents should be re-suspended in nuclease-free ddH₂O. RNase free techniques should be used when handling reagents and when making the injection mix.

Table 2. Worm Lysis Buffer Recipe. Proteinase K should be added fresh before each use.

Table 3. PCR Mastermix. The PCR mastermix should be mixed well by pipetting until the solution is homogeneous. 21 µL of the PCR mastermix should be added to clean PCR tubes, and then 4 µL of individual worm lysate should be added to properly labeled tubes.

Table 4. Restriction Enzyme Mastermix. The restriction enzyme mastermix should be mixed well by pipetting until the solution is homogeneous. 10 µL of the restriction enzyme mastermix should be added to 10 µL of cleaned PCR product