CRISPR-Cas9-based repeat depletion for high-throughput genotyping of complex plant genomes

Abstract

High-throughput genotyping enables the large-scale analysis of genetic diversity in population genomics and genome-wide association studies that combine the genotypic and phenotypic characterization of large collections of accessions. Sequencing-based approaches for genotyping are progressively replacing traditional genotyping methods due to the lower ascertainment bias. However, genome-wide genotyping based on sequencing becomes expensive in species with large genomes and a high proportion of repetitive DNA. Here we describe the use of CRISPR-Cas9 technology to deplete repetitive elements in the 3.76-Gb genome of lentil (Lens culinaris), 84% consisting of repeats, thus concentrating the sequencing data on coding and regulatory regions (single-copy regions). We designed a custom set of 566,766 gRNAs targeting 2.9 Gbp of repeats and excluding repetitive regions overlapping annotated genes and putative regulatory elements based on ATAC-seq data. The novel depletion method removed ~40% of reads mapping to repeats, increasing those mapping to single-copy regions by ~2.6-fold. When analyzing 25 million fragments, this repeat-to-single-copy shift in the sequencing data increased the number of genotyped bases of ~10-fold compared to nondepleted libraries. In the same condition, we were also able to identify ~12-fold more genetic variants in the single-copy regions and increased the genotyping accuracy by rescuing thousands of heterozygous variants that otherwise would be missed due to low coverage. The method performed similarly regardless of the multiplexing level, type of library or genotypes, including different cultivars and a closely-related species (L. orientalis). Our results demonstrated that CRISPR-Cas9-driven repeat depletion focuses sequencing data on meaningful genomic regions, thus improving high-density and genome-wide genotyping in large and repetitive genomes.

guidedNOMe-seq quantifies chromatin states at single allele resolution for hundreds of custom regions in parallel.

“…In our study we used a pool of 6000 sgRNAs to perform target enrichment. Recently, depletion experiments were successfully done with a pool of ~ 500 000 sgRNAs in a single Cas9 digestion experiment (Rossato et al 2023).This indicates that the number of sgRNAs and therefore ROI can be signi cantly increased for guided-NOMe-seq as well.…”

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

“…After cleavage, the plant’s (rice) 16S rRNA fragments are not amplified in the second PCR and are then excluded from the sequencing. In order to eliminate repetitive sequences from a genome that is particularly rich in repetitive elements, such as that of lentils, the target specificity of the CRISPR/Cas system combined with a set of over 500,000 gRNAs was used and allowed to exclude 40% of the reads mapping on repeats from sequencing and to enhance the number of reads mapping on unique regions by more than twice as much [ 104 ]. Using such methodologies allows us to focus on relevant genomic regions and to increase genotyping accuracy.…”

Enhancing biodiversity monitoring efficiency through CRISPR-driven depletion and enrichment of aquatic environmental DNA

“…DASH works by cleaving target DNA in a sequencing library so that only non-targeted sequences with adapters at both ends remain to be sequenced. Rossato et al (2023) demonstrated the effectiveness of using the DASH method by using 566,766 sgRNAs to deplete repetitive elements in a lentil genome to improve genotyping methods. DASH-depletion resulted in a 37.7%-41.2% reduction of repetitive DNA sequences, with an increase of up to 160% in target DNA reads sequenced.…”

“…These methods both have relatively short lab protocols as the most timeconsuming step is a two-hour incubation. As with most initially expensive technologies, the cost of CRISPR-Cas assays can reduce dramatically when used in repeated monitoring (Rossato et al, 2023).…”