Main menu

CRISPR-Cas9 enrichment and long read sequencing for fine mapping in plants


Background

Genomic methods for identifying causative variants for trait loci applicable to a wide range of germplasm are required for plant biologists and breeders to understand the genetic control of trait variation.

Results

We implemented Cas9-targeted sequencing for fine-mapping in apple, a method combining CRISPR-Cas9 targeted cleavage of a region of interest, followed by enrichment and long-read sequencing using the Oxford Nanopore Technology (ONT). We demonstrated the capability of this methodology to specifically cleave and enrich a plant genomic locus spanning 8 kb. The repeated mini-satellite motif located upstream of the Malus × domestica (apple) MYB10 transcription factor gene, causing red fruit colouration when present in a heterozygous state, was our exemplar to demonstrate the efficiency of this method: it contains a genomic region with a long structural variant normally ignored by short-read sequencing technologies

Cleavage specificity of the guide RNAs was demonstrated using polymerase chain reaction products, before using them to specify cleavage of high molecular weight apple DNA. An enriched library was subsequently prepared and sequenced using an ONT MinION flow cell (R.9.4.1). Of the 7,056 ONT reads base-called using both Albacore2 (v2.3.4) and Guppy (v3.2.4), with a median length of 9.78 and 9.89 kb, respectively, 85.35 and 91.38%, aligned to the reference apple genome. Of the aligned reads, 2.98 and 3.04% were on-target with read depths of 180 × and 196 × for Albacore2 and Guppy, respectively, and only five genomic loci were off-target with read depth greater than 25 × , which demonstrated the efficiency of the enrichment method and specificity of the CRISPR-Cas9 cleavage.

Conclusions

We demonstrated that this method can isolate and resolve single-nucleotide and structural variants at the haplotype level in plant genomic regions. The combination of CRISPR-Cas9 target enrichment and ONT sequencing provides a more efficient technology for fine-mapping loci than genome-walking approaches.

Authors: Elena López-Girona, Marcus W. Davy, Nick W. Albert, Elena Hilario, Maia E. M. Smart, Chris Kirk, Susan J. Thomson & David Chagné

入门指南

购买 MinION 启动包 Nanopore 商城 测序服务提供商 全球代理商

纳米孔技术

订阅 Nanopore 更新 资源库及发表刊物 什么是 Nanopore 社区

关于 Oxford Nanopore

新闻 公司历程 可持续发展 领导团队 媒体资源和联系方式 投资者 合作者 在 Oxford Nanopore 工作 职位空缺 商业信息 BSI 27001 accreditationBSI 90001 accreditationBSI mark of trust
Chinese flag