An efficient CRISPR-Cas9 enrichment sequencing strategy for characterizing complex and highly duplicated genomic regions. A case study in the Prunus salicina LG3-MYB10 genes cluster

Abstract

Background Genome complexity is largely linked to diversification and crop innovation. Examples of regions with duplicated genes with relevant roles in agricultural traits are found in many crops. In both duplicated and non-duplicated genes, much of the variability in agronomic traits is caused by large as well as small and middle scale structural variants (SVs), which highlights the relevance of the identification and characterization of complex variability between genomes for plant breeding. Results Here we improve and demonstrate the use of CRISPR-Cas9 enrichment combined with long-read sequencing technology to resolve the MYB10 region in the linkage group 3 (LG3) of Japanese plum (Prunus salicina). This region, which has a length from 90 to 271 kb according to the P. salicina genomes available, is associated with fruit color variability in Prunus species. We demonstrate the high complexity of this region, with homology levels between Japanese plum varieties comparable to those between Prunus species. We cleaved MYB10 genes in five plum varieties using the Cas9 enzyme guided by a pool of crRNAs. The barcoded fragments were then pooled and sequenced in a single MinION Oxford Nanopore Technologies (ONT) run, yielding 194 Mb of sequence. The enrichment was confirmed by aligning the long reads to the plum reference genomes, with a mean read on-target value of 4.5% and a depth per sample of 11.9x. From the alignment, 3261 SNPs and 287 SVs were called and phased. A de novo assembly was constructed for each variety, which also allowed detection, at the haplotype level, of the variability in this region. Conclusions CRISPR-Cas9 enrichment is a versatile and powerful tool for long-read targeted sequencing even on highly duplicated and/or polymorphic genomic regions, being especially useful when a reference genome is not available. Potential uses of this methodology as well as its limitations are further discussed.

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

“…This is particularly beneficial in the case of plants with large and complex genomes, which limit the ability to generate multiple whole-genome assemblies in a large set of individuals. The reconstructed ROI can replace poor-quality assemblies in the reference genome and can fill gaps, as already attempted in the Japanese plum ( Prunus salicina ) [ 22 ]. We found that the availability of more precise reconstructions allows reads to be mapped more accurately in cultivars/varieties of interest and facilitates downstream variant calling.…”

“…For the same reason, the size of the enriched region is usually limited to <10–15 kbp [ 17 ]. More recently, CRISPR/Cas9-based enrichment has emerged as a promising approach that is not dependent on amplification, allowing the enrichment of targets up to 80 kbp [ 19 , 20 , 21 , 22 , 23 ]. The enrichment of longer targets (up to 100 kbp) is possible using the ONT platform if shorter non-target fragments that compete for sequencing are depleted [ 24 ].…”

“…However, this is a relatively new approach and the largest sub-ROI that has been sequenced thus far is 25 kbp [ 25 , 26 ]. Even so, this approach was shown to resolve SNVs and SVs at the haplotype level, which was not possible with short reads [ 22 , 23 ]. The validity and accuracy of the method for de novo assembly of large ROIs compared to traditional long-read whole-genome sequencing (WGS) is not yet clear.…”

Spontaneous, Artificial, and Genome Editing-Mediated Mutations in Prunus

“…Another application of CRISPR/Cas9 enrichment has been through the combination with long-read sequencing technology in order to solve complex genomic regions. This has been successfully applied to the MYB10 region on chromosome 3, which is significantly linked to fruit color variability in Prunus species [ 136 ]. In this work, it was possible to apply this technology to cleave MYB10 genes of five plum varieties ( Prunus salicina L.) proving to be a useful tool for long-read targeted sequencing when the reference genome was not available.…”

Cas9-Mediated Nanopore Sequencing Enables Precise Characterization of Structural Variants in CCM Genes

“…However, both ends of the target region need to be known. A combination of both approaches can be applied to larger regions [ 22 ].…”