CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.

This technique is beneficial for introducing precise mutations into C.elegans by CRISPR/Cas9 genome editing within a short time frame. This technique provides an efficient, streamlined manner for editing the C.elegans genome, as the use of a Co-CRISPR marker greatly improves the probability of identifying positively edited worms. Be sure to practice performing the microinjections, and to inject both of the gonad arms of as many worms as possible to increase the chances of obtaining the desired edits.

Demonstrating the procedure will be Amy Smith, Mary Bergwell, Danita Mathew, Ellie Smith, and Carter Dierlam, who are all undergraduate students from my laboratory. To prepare young adult worms for injection, pick L2, L3 stage C.elegans onto a fresh bacterial lawn on an MYOB plate and incubate the plate at 20 degrees Celsius overnight. The next morning, prepare the injection mix as outlined in the table in sterile nuclease-free tubes and mix by pipetting.

Incubate the injection mix at 37 degrees Celsius for 15 minutes to assemble the ribonucleoprotein complexes before spinning down the mix by centrifugation. Next, use a dissecting microscope to pick about 30 young adult worms with fewer than 10 embryos in the uterus, and inject both gonad arms of each worm with a few picoliters of injection mix per arm. After microinjection, use a recovery apparatus to gently mouth pipette injected worms from the injection pad onto a 60 millimeter MYOB plate seeded with OP50 E.coli.

Allow the injected worms to recover for one hour at room temperature. At the end of the recovery period, use a platinum wire pick to transfer each injected worm onto a single seeded 35 millimeter MYOB agar plate, and allow the worms to lay eggs at 20 degrees Celsius. After 24 hours, transfer the injected worms to new individual 35 millimeter plates.

For three to four days after the injection, use a dissecting microscope to identify plates that have F1 progeny exhibiting roller phenotypes, as indicated by the body of the animal rotating around its long axis as the animals move in a circular pattern, and dumpy phenotypes, as evidenced by a shorter and stouter physiology compared to control animals at the same developmental stage. Transfer 50 to 100 F1 roller worms to their own individual plates and allow the worms to lay eggs for one to two days. After one to two days of F2 worm production, transfer each single F1 roller mother from a numbered plate into its corresponding numbered PCR tube containing 2.5 microliters of lysis buffer supplemented with a 1:100 dilution of 20 milligrams per milliliter proteinase K.Freeze the tubes at minus 30 degrees Celsius for 20 minutes before lysing worms in a thermocycler, as indicated.

At the end of the cycle, add 12.5 microliters of 2X PCR mix containing deoxynucleoside triphosphates, DNA polymerase, magnesium chloride, and loading dye, one microliter of 10 micromolar forward primer, one microliter of 10 micromolar reverse primer, and enough sterile water to bring the reaction mixture to 25 microliters per each lysed worm sample. Then run the samples on the thermocycler as indicated. To screen for edits less than 50 base pairs in length, transfer 10 microliters of each PCR reaction to a new tube and add five microliters of the restriction digestion master mix to each tube.

Incubate the digestion reactions at 37 degrees Celsius for two hours. At the end of the incubation, heat and activate the digestions with a 10 to 15 minute incubation at 65 degrees Celsius. Then load the entire reaction from each tube into a single well of a 1%to 2%agarose gel and run the gel at 110 milliamps until proper band separation is achieved.

At the end of the run, visualize agarose gels under UV light to detect the DNA band sizes. Positively edited worms will display an extra band of DNA at the expected size due to editing using the repair template, carrying the restriction site at the desired locus within the worm genome. Pick eight to 12 wild type-looking non-rolling F2 worms from the respective positively edited F1 roller worm plates and allow the worms to lay eggs and to produce progeny for one to two days.

Then identify the homozygous worm lines as demonstrated for positively edited worms and analyze the sequencing results using sequence analysis software to confirm the presence of the edit according to standard protocols. Here, the genomic DNA and the designed CRISPR RNA and repair template for the C.elegans rbm-3.2 gene can be observed. In this representative analysis, seven out of 73 F1 worms were found to be positive for the edit.

Unedited worms displayed the single DNA fragment of 445 base pairs upon EcoRI digestion, while worms carrying a premature stop codon in the rbm-3.2 gene exhibited two fragments at 265 and 166 base pairs upon EcoRI digestion. Heterozygous edited worms displayed three fragments upon EcoRI digestion. Six out of 12 F2 worms from the identified positive F1 heterozygotes were found to be homozygous for the edit of interest.

The genomic DNA of an identified homozygous edited worm line could then be used to set up a DNA sequencing reaction, which confirmed the presence of the edit in its homozygous state. Take care that the editing reagents and screening primers are designed correctly, and that the injection mix is prepared properly. An accurate microinjection is also critical.

Following this procedure, phenotypic validation of the edit, if applicable, and gene and protein expression analysis may be performed to verify the accuracy of the genome editing.