CRISPR/Cas9-Mediated Enrichment Coupled to Nanopore Sequencing Provides a Valuable Tool for the Precise Reconstruction of Large Genomic Target Regions

Abstract

Complete and accurate identification of genetic variants associated with specific phenotypes can be challenging when there is a high level of genomic divergence between individuals in a study and the corresponding reference genome. We have applied the Cas9-mediated enrichment coupled to nanopore sequencing to perform a targeted de novo assembly and accurately reconstruct a genomic region of interest. This approach was used to reconstruct a 250-kbp target region on chromosome 5 of the common bean genome (Phaseolus vulgaris) associated with the shattering phenotype. Comparing a non-shattering cultivar (Midas) with the reference genome revealed many single-nucleotide variants and structural variants in this region. We cut five 50-kbp tiled sub-regions of Midas genomic DNA using Cas9, followed by sequencing on a MinION device and de novo assembly, generating a single contig spanning the whole 250-kbp region. This assembly increased the number of Illumina reads mapping to genes in the region, improving their genotypability for downstream analysis. The Cas9 tiling approach for target enrichment and sequencing is a valuable alternative to whole-genome sequencing for the assembly of ultra-long regions of interest, improving the accuracy of downstream genotype–phenotype association analysis.

Bridging the gap: a prospective trial comparing programmable targeted long-read sequencing and short-read genome sequencing for genetic diagnosis of cerebellar ataxia

“…Reliably achieving these targets may be challenging given the competing need to minimize the target panel for maximum enrichment versus increasing panel size to reduce nanopore destruction due to polarity reversal associated with off-target rejection. Additional enrichment strategies, including CRISPR/Cas9, have been proposed with LR-AS, or indeed as an alternative enrichment protocol (Mizuguchi et al 2021;Lopatriello et al 2023;Leitao et al 2024). However, in the context of diagnostic application these approaches are bespoke, require considerable optimization and are limited in the number of targets that can be successfully multiplexed.…”

Not Only Editing: A Cas-Cade of CRISPR/Cas-Based Tools for Functional Genomics in Plants and Animals

“…In this case, the ROI is divided into smaller overlapping sub-regions (sub-ROI) of lengths of up to 25 kb and are enriched and sequenced separately. Finaly, in a study proposed by Lopatriello et al, the Cas9-tiling approach allowed the de novo assembly and identification of SVs of a 250-kb region on chromosome Pv05 of Phaseolus vulgaris [ 126 ].…”