Making point mutations using CRISPR/Cas9 and the pha-1 co-CRISPR marker
To generate point mutations, we currently favor the use of single-strand oligo DNA (ssODN) repair templates (Zhao et al) in combination with the use of pha-1 rescue as a co-CRISPR marker, as described by Ward, J.. In this approach, a guide RNA and ssODN repair template that convert a temperature sensitive pha-1(e2123) allele into a wild-type pha-1 allele are co-injected with the sgRNA and repair template that generate the mutation of interest. The e2123 mutation causes a temperature sensitive lethal phenotype at 25ºC. F1 animals that survive are likely enriched for your desired genome engineering. A limitation of this selection method is that it requires a pha-1(2123) background.
Selection of the sgRNA target sequence
For oligonucleotide mediated repair, you will want the sgRNA target sequence to be close to the site you want to mutate (<30 bp) in order to minimize the size of the oligo that needs to be ordered.
Designing the repair template
Design an oligo nucleotide as follows:
- Incorporate the desired mutation(s).
- Incorporate mutations in the sgRNA target site to prevent recutting after repair. Ideally, mutate one of the Gs in the PAM sequence. If that is not feasible, incorporate silent mutations close to the 3'-end of the 20 bp recognition sequence.
- Incorporate silent mutations that create a unique primer binding site, to enable detection of the desired change by PCR. In our hands, primers with several mismatches, even of the 3'-most nucleotide, still yield PCR product. Hence, we try to mutate 4-5 basepairs, including the 3'-most basepair if possible. Ideally, the changed sites are near the desired mutation, especially if the sgRNA recognition sequence is futher away from the desired mutation site. However, the mutations can be introduced anywhere, including overlapping the sgRNA target site. This would simultaneously prevent recutting after repair.
- Use 35-40 bp homology arms flanking the first and last mutation.
- Sense oligos with respect to the strand of the gene have been reported to work best.
Example of introducing a mutation. In this example, a Ser to Ala change is desired. A sgRNA target site is located nearby. The ssODN repair template contains the desired change, plus a number of silent changes that simultaneously alter the target site so it is no longer recognized after repair, and serve as a primer binding site that can be used to detect a successful genome edit event. Not indicated is a second primer needed for detection, which can be chosen anywhere in the next 300 - 2000 nucleotides.
Primers to design
For the detection and sequencing of a successfully engineered point mutation, 4 primers need to be designed (see image above):
- Primer F1 anneals to the left of the mutated site, and is used in combination with primer R1 to amplify a small region to be sequenced to confirm the mutation. Primer F1 also serves as the sequencing primer, so should not be placed too far away from the mutated site (generally < 250 bp away should result in a good sequence quality near the mutated site).
- Primer F2 should overlap the mutation site, and should only anneal to the wild-type sequence.
- Primer F3 should overlap the mutation site, and should only anneal to the mutated sequence (the forward detection primer in the template design figure).
- Primer R1 is the reverse primer for all forward primers.
Primers F2 and F3 are used to determine the genotype of (candidate) edited animals:
- Animals that are homozygous wild-type will only show an amplicon using F2/R1.
- Animals that are homozygous mutant will only show an amplicon using F3/R1.
- Heterozygous animals will yield a PCR product with both primer sets.
Inject the following mixture into 20-40 pha-1(2123) adults (we currently do not use a fluorescent co-injection marker):
- 50 ng/µl ssODN repair template carrying your desired mutation
- 50 ng/µl sgRNA construct targeting your gene of interest
- 50 ng/µl ssODN repair template to correct the pha-1(e2123) mutation.
- 60 ng/µl pJW1285 - Peft-3::Cas9 and sgRNA construct targeting pha-1 (Jordan Ward, Addgene plasmid #61252)
Following the injection, pool 3 - 5 P0s per NGM plate and incubate plates at 25ºC. Alternatively, worms can be singled into individual NGM plates to facilitate distinction of independent events.
The sequence of the pha-1(e2123) ssODN:
The sequence of the pha-1 targeting sgRNA:
Detection of mutation by PCR
- Wait for surviving F1 worms to appear on the P0 plates. Worms only survive at 25ºC if at least one of the pha-1(2123) copies reverts to WT by CRISPR-mediated repair.
- Pick rescued animals to individual plates, and allow these to produce progeny overnight.
- Lyse the F1 animals, and use the F3/R1 primer pair to detect the mutation.
- Recover the mutant strain from the F2 progeny of F1 animals that show the desired mutation by PCR. Use primer sets F2/R1 and F3/R1 to identify homozygous mutant animals (this may not be possible if the mutation is lethal).
- Use primer set F1/R1 to amplify the region containing the mutation and confirm by sequencing. Preferably this is done on a homozygous mutant animal. If this is not possible, the mutation can be characterized by sequencing a heterozygous animal, as each mutated site should result in a double peak.
- To remove the single copy of pha-1(e2123) that might remain, either keep the worms at 25ºC for a few generations or backcross and sequence the pha-1 locus.