Protocols: Testing gRNA cleavage efficiency

Embryonic PCR Protocol

 

Embryonic PCRs are very valuable in determining the cleavage efficiency of a pair of gRNAs as well as HDR efficiency. These reactions can be done on embryos 24 hours after injections for rapid feedback on the likelihood a particular strategy will work.

 

Designing Primers

  • Testing a pair of gRNAs: Design oligos flanking the two cut sites (i.e. spanning a deletion created by cleavage at both sites). We generally pick priming sites ~200-300 bp outside of the target sites such that 400-600 bp band will indicated cleavage at both sites.

  • Testing HDR efficiency: Design one oligo specific to the marker, insertion, or allele used in your HDR strategy. For example, when using pHD-DsRed-attP, we use primers specific to the 3xP3-DsRed-SV40 portion of the vector. Design a second oligo specific to the target locus but outside of the homology arms such that the PCR is specific for integration at the target locus.  For strategies involving two distinct homology arms, as with pHD-DsRed-attP, we use two separate PCR reactions to test each side of the intended HDR event.

 

Preparing embryonic genomic DNA

1. Pick individual embryos
Transfer individual embryos 24 hours after injection into 0.2 mL PCR tubes. Place them on the side of the tube. Typically we use 16-32 embryos to an idea of the efficacy of a particular strategy of pair of gRNAs.
 
2. Prepare embryo-smashing buffer
 
1X embryo smashing buffer recipe:
         Tris-HCl (pH 8.2): 10 mM
         NaCl: 25 mM
         EDTA: 1 mM
         Triton x100: 0.2% (v/v)
 
Just before use, dilute Proteinase K into buffer at 200 ug/mL
 
3. Smash each embryo
Using the side of a P200 tip, smash the embryo against the side of the PCR tube. Then add 20 uL of smashing buffer to the PCR tube making sure to rinse the area where the embryo was smashed to sufficiently suspend the embryo debris.
 
4. Incubate
Incubate the tube at 37C for 40 minutes and then heat inactivate the Proteinase K at 95C for 5 minutes.
 
5. PCR
Use 1 uL of smash prep per 20 uL PCR reaction.