Detection of disease causing repeats by multi-locus CRISPR-Cas enrichment and nanopore sequencing

With the maturation of long-read sequencing technologies, the applications of genome diagnostic tests in patient care are expanding. A promising application is the sequencing of clinically relevant repeat structures in disorders such as ALS, Huntington's disease, fragile X syndrome, and spinocerebellar ataxias. Increasing repeat units within such a repeat locus are indicative of disease severity and age of onset, and are therefore of clinical importance. Due to the repetitive nature and length (up to several kilobases) it is hard to determine the exact structure of repeats with traditional sequencing technologies. Long-read nanopore sequencing has the potential to overcome this problem by sequencing the entire disease locus including the full (expanded) repetitive region and associated epigenetic modifications.
We present our work on the development and testing of a cost-effective and comprehensive diagnostic test that targets clinically relevant repeat structures in one experiment. The test is based on multiplexed sequence enrichment of ten repeat loci using CRISPR-Cas technology combined with Oxford Nanopore sequencing. We used this approach to sequence controls and patients and demonstrate that our approach is able to effectively enrich and sequence clinically relevant genomic repeat structures. In comparison to our current diagnostic assays we show that the number of repeat units within these loci can be detected with high accuracy.