Model Generation Frequently Asked Questions
Design & Planning
Q: Why are small KI models created with oligo's and larger KI models created with plasmid donors?
A: Small knock-ins, such as a point mutation or a tag insertion may be accomplished by quickly synthetizing donor oligonucleotides up to 200 base pairs, which act as a donor for homology directed repair after CRISPR/Cas9 system generates double strand breaks in the genome. Larger KI is more efficient with a donor with longer homologous sequence and hence requires a plasmid donor.
Q: What does JAX do to minimize off-target events?
A: Guides used in our CRISPR/Cas9 strategy are chosen to optimize efficiency while minimizing off-target effects. Factors taken into consideration include: sequence matches elsewhere in the genome other than the intended target site, the use of nickase strategy, purity and concentration of the CRISPR/Cas9 reagents, the use of circularized donor plasmid, etc. If the off-target effects are of concern for a particular project, we can address the concern by performing off-target analysis.
Q: How do you reduce the risk of mosaicism? Any special screening?
A: Founder mice from a CRISPR/Cas9 project could be mosaic. Sequencing data of the alleles can be further decomposed using a software algorithm to characterize the nature of the mosaicism. It is possible to predict the different resulting alleles in founder mice and subsequently pick the animals most likely to be successful for N1 generation. However, it is important to note that due to mosaicism, the alleles seen from a tail snip or ear punch of founder mice may not necessarily be the same alleles found in sperm or oocytes that can be transmitted from founder to N1, so some alleles may be lost in germline transmission confirmation breeding. Thus, we recommend sending multiple founder animals (3-5) for N1 breeding.
Q. Can JAX provide back sequence(s) of the specific guide RNA used to create the model?
A: It is included in the Project Plan.
Q: How are embryos, injections and transfers performed?
A: Embryos are isolated from pregnant females in the morning on the day of injection to ensure that they are at the 1-cell stage. CRISPR components (Cas9 mRNA, sgRNA, and any necessary DNA oligo's or plasmids) are injected into the male pro-nucleus.
Q: Does the genotyping of the founders include evaluation of other mutations that may have been introduced in/around the site of the intended mutation site?
A: Yes, we usually sequence ~400 bp region around any guide target sequence which should cover projects involving Indels and point mutations. If a plasmid donor is used, then LR-PCR would evaluate overall integrity of the targeted locus and the region around the end of homology arms and the insertion site should be sequenced.
Q: Does the genotyping also involve evaluation for mosaicism?
A: We will genotype the founder mice by direct sequencing of PCR product. Analysis of sequencing results usually allows us to estimate relative frequency of the WT and modified alleles.
Q: Are there ways to skew the deletions versus the indels at the individual sgRNA sites?
A: We are still figuring out what conditions favor large deletion over Indels.
Q: Can I get sequence files of the indel genotyping sequence for my project?
A: We will send you raw sequencing data.
Q: Are the founders and N1 both homozygous?
A: It is possible to generate founder mice homozygous for a particular allele. However, seemingly homozygous mice based on PCR genotyping can have mosaicism in different tissues including germline cells.
After breeding to WT animals, N1 would be heterozygous for the targeted allele. Homozygous mice can be generated in the N2 generation.
Q: Are the frameshift mutations causing a premature stop? In other words, how sure are you that we have created a KO?
A: There are multiple out-of-frame stop codons in the downstream coding exons, which would cause premature termination of translation in frameshifting Indel mutants.
Q: How do you choose the founders to breed forward?
A: Recommendation was based on the frequency of frameshifting Indel alleles, but eventually it is your choice.
Q: For N1 generation, why should we cross breed wild type mice with our founder? I wonder can't we just breed a pair of founders followed by keep inbreeding sister and brother or backcross? Or breed with single KO mice strain?
A: In CRISPR-generated alleles, frameshifting Indel mutations will be created through NHEJ-mediated repair system. This method is prone to generate multiple different Indel alleles of the targeted gene even in a single founder animal. Due to this mosaicism, we recommend any founder is bred to a WT mouse to confirm germline transmission and separate individual alleles in N1 mice. This also reduces potential risk of off-target effect.