Availability of long read DNA sequencing provides a rigorous and reproducible method to characterize models made through modern CRISPR methods or even those made through traditional transgenic/gene targeting methods. Common approaches such as PCR based genotyping are powerful, but may not always pick up every class of unexpected mutation. In contrast, NexGen approaches such as whole genome sequencing using PacBio or ONT technologies can provide both local structural information, along with small mutational analysis.
If you’re planning on publishing your edited murine models, cell lines, etc., we recommend our new Allele Auditing Service, where we confirm your allele of interest using long read technologies followed by careful inspection of your locus. The AGEL will coordinate your sample intake, DNA prep, library preparation, sequencing via PacBio Sequel II, and bioinformatic analysis for $2500. In return, you will have access to the structural analysis of your edited region, robust molecular evidence that the locus meets expectations, as well as identification of any subtle mutations you may not have anticipated.
In addition to providing increased rigor to your work, the approach offers potential for unexpected new tools. Using an approach, we refer to as Allele Salvaging, laboratories can now take advantage of unexpected mutations that arise from off target gene editing events. In one recent manuscript (https://pubmed.ncbi.nlm.nih.gov/33480436/) arising from this approach, a laboratory was able to interrogate three novel alleles at the Ahr locus. Two of which arose through unexpected editing events that were only revealed after careful Allele Auditing.