Resolving the CES1 Genomic Locus with Cas9-Directed Targeted Long-Read Sequencing for Precision Pharmacogenomics

Carboxylesterase 1 (CES1) is the primary hepatic hydrolase in humans, crucial for the metabolism of ester-containing drugs and endogenous lipids. However, the CES1 genomic region is difficult to resolve because of adjacent highly homologous pseudogenes and the presence of large structural variants. These complexities often cause read misalignment and inaccurate variant calling with conventional short-read sequencing, hindering reliable pharmacogenomic analyses. To overcome these limitations, we employed an optimized, PCR-free Oxford Nanopore Technologies (ONT) sequencing method, Cas9directed targeted sequencing (nCATS), to characterize the targeted region of up to 76 kb, including CES1, CES1P1 or CES1A2, and their intergenic regions. This approach uses Cas9 to selectively enrich and sequence long native DNA fragments, while avoiding amplification-induced artefacts. Long-read sequencing was performed in 23 human blood samples and the HepG2 hepatoblastoma cell line enabling high-resolution mapping to the CES1 locus. We uncover five previously unrecognized main CES1 haplotypes and report that many single nucleotide variants (SNVs) in public databases are likely artefacts caused by short-read misalignment. Additionally, we identify long inverted repeats (LIRs) flanking a fragile genomic site within the region, which may form DNA hairpins and contribute to structural plasticity at the locus. This study demonstrates the utility of long-read sequencing for resolving complex genomic regions such as CES1, allowing comprehensive detection of structural variants and haplotype-resolved SNVs. Our findings provide improved reference sequences and deeper insight into CES1 diversity, with significant implications for future pharmacogenetic research and the development of personalized treatment strategies involving CES1-metabolized medications.