Source: Prior, H., et al. A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins. J. Vis. Exp. (2018).
This video describes a technique to rapidly screen a single nematode for a genetic marker by PCR screening. In the example protocol, we screen for CRISPR-based genome editing.
The following protocol is an excerpt from Prior et al, A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins, J. Vis. Exp. (2018).
Single Worm PCR and Genotyping
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.
Reagent | [Final] |
KCl | 50 mM |
Tris-HCl pH 8.3 | 10 mM |
MgCl2 | 2.5 mM |
NP-40 | 0.45% |
Tween-20 | 0.45% |
Proteinase K | 1 mg/mL |
Table 1. Worm Lysis Buffer Recipe. Proteinase K should be added fresh before each use.
Reagent | 1x | 24x |
2X Q5 Mastermix | 12.5 μL | 300 μL |
Primer F1 (10 μM) | 1.25 μL | 30 μL |
Primer R1 (10 μM) | 1.25 μL | 30 μL |
Worm Lysis | 4 μL | —- |
ddH2O | 6 μL | 144 μL |
Final Volume | 25 μL | 504 μL |
Table 2. 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.
Reagent | 1x | 24x |
10X Enzyme Buffer | 2 μL | 48 μL |
Cleaned PCR Reaction | 10 μL | —- |
ddH2O | 7 μL | 168 μL |
Restriction Enzyme (10 U/μL) | 1 μL | 24 μL |
Final Volume | 20 μL | 240 μL |
Table 3. 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.
Nuclease-free water | Synthego Inc. | provided with the sgRNA kit EZ kit | |
KCl | Sigma | P5405 | |
DNA Clean & Concentrator | Zymo Research | D4004 | |
Zymoclean Gel DNA Recovery Kit | Zymo Research | D4002 | |
Q5 Hot Start High-Fidelity 2X Master Mix | New England Biolabs | M0494L | |
Proteinase K | Sigma | P2308 | |
MgCl2 | Sigma | M2393 | |
NP-40 | Sigma | 74385 | |
Tween-20 | Fisher Scientific | BP337-100 | |
RNaseZap Decontamination Solution | Fisher Scientific | AM9780 |