RT Journal
A1 Rossato, Marzia
A1 Marcolungo, Luca
A1 De Antoni, Luca
A1 Lopatriello, Giulia
A1 Bellucci, Elisa
A1 Cortinovis, Gaia
A1 Frascarelli, Giulia
A1 Nanni, Laura
A1 Bitocchi, Elena
A1 Di Vittori, Valerio
A1 Vincenzi, Leonardo
A1 Lucchini, Filippo
A1 Bett, Kirstin E.
A1 Ramsay, Larissa
A1 Konkin, David James
A1 Delledonne, Massimo
A1 Papa, Roberto
T1 CRISPR-Cas9-based repeat depletion for high-throughput genotyping of complex plant genomes
JF Genome Research 
JO Genome Research 
YR 2023 
FD May 01 
VO 33 
IS 5 
SP 787 
OP 797 
DO 10.1101/gr.277628.122 
UL http://genome.cshlp.org/content/33/5/787.abstract 
AB 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 because of 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 because of 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 showed that CRISPR-Cas9-driven repeat depletion focuses sequencing data on single-copy regions, thus improving high-density and genome-wide genotyping in large and repetitive genomes.