Guide RNA Testing in Mouse Zygotes

General Description

The genome editing efficiency of up to two guide RNAs selected by the core as part of a project design or two guide RNAs selected by an investigator and approved by the core will be tested in mouse embryos.

What Will Happen

  1. The user investigator will initiate a project in iLabs. An account must be provided at this time.
  2. The core will use web-based applications to assess predicted guide RNA cutting efficiency.
  3. The core will design a PCR-based assay to detect indels resulting from NHEJ. A PCR protocol will be optimized using wild-type DNA.
  4. The core will have the guide RNA synthesized by an approved vendor. Lab produced guide RNAs will not be tested.
  5. The guide RNA will be checked for concentration and degradation after resuspension.
  6. The guide RNA and Cas9 protein will be complexed into RNPs and an electroporation mix will be prepared.
  7. 40-50 C57BL/6J embryos collected from superovulated females will be electroporated and subsequently cultured to the blastocyst stage. Embryos from other strains can be tested if necessary. Please contact the GERM Core to discuss the necessary steps involved to use other strains.
  8. Blastocyst DNA will be isolated.
  9. PCR will be performed on blastocyst and the resulting PCR products Sanger sequenced.
  10. ICE analysis (Synthego) will deconvolute sequencing traces for each blastocyst to identify blastocysts with indel alleles and the percentage of indel alleles detected per blastocyst.

What to Expected

  1. Some embryos will die during culture or enough DNA may not be isolated for PCR. We will guarantee a minimum of 12 blastocysts that produce a PCR product. Additional electroporations will be performed if necessary.
  2. Genome editing in mouse zygotes often results in mosaicism. Thus, multiple indel alleles are likely to be detected within a single blastocyst. Efficiency of the guide will be determined by the percentage of alleles with an indel.
  3. To produce mouse lines a guide RNA should have the following efficiencies:
    • Projects requiring NHEJ (e.g. knockout allele production): At least 60% of blastocysts with a detected indel allele
    • Projects requiring HDR (e.g. point mutations): At least 75% of blastocysts with a detected indel allele.