Poster presented at the 2023 Advances in Genome Biology and Technology (AGBT) Precision Health Meeting.
Gabriel E. Rech1, Chrystal Snow2 and Chia-Lin Wei1,2.
1The Jackson Laboratory for Genomic Medicine, Farmington, CT, 06032, USA. 2The Jackson Laboratory, Bar Harbor, ME, 04609, USA.
The ability to modify genomes of model organisms using a wide range of genetic manipulation methods has provided us tools to study disease mechanisms and devise therapeutic strategies. Yet, the approaches to determine transgene integration or examine the modified loci are currently limited. The Jackson Laboratory is the world's largest source of genetically defined mice strains, sourcing more than 8,000 strains worldwide. The traditional and gold standard method for validating mouse models has been PCR based assays and Sanger sequencing. Despite its robustness, Sanger sequencing method is not highly sensitive on identifying novel low-frequency genomic alterations and can struggle to accurately characterize large and complex regions of the genomes. We have developed robust targeted long-read sequencing platforms for the identification and characterization of genome editing events in host genomes. Our established platform applies a CRISPR-Cas9 targeted approach, combined with long-read nanopore sequencing, to enhance sequencing depth at specific loci of interest. This platform provides an end-to-end workflow to fine map vector/transgene integration sites and their associated genomic aberration. The entire workflow has been optimized with a wide range of sample types. Because the robustness of the capture reactions and the simplicity of the nanopore sequencing operation, the process can be easily automated and is scalable to achieve high throughput and consistency. We expect that this platform will be valuable for vector integration assessment and reveal functional insight in the frequency, complexity, impacts and clonality of the integration events.
