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. 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. 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.

中文翻译：

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CRISPR-Cas9 富集和长读长测序，用于植物精细定位。

植物生物学家和育种者需要采用基因组方法来识别适用于多种种质的性状基因座的致病变异，以了解性状变异的遗传控制。我们在苹果中实施了用于精细定位的 Cas9 靶向测序，该方法结合了 CRISPR-Cas9 对感兴趣区域的靶向切割，然后使用牛津纳米孔技术 (ONT) 进行富集和长读长测序。我们证明了这种方法能够特异性切割和富集跨度为 8 kb 的植物基因组位点。重复的小卫星基序位于 Malus × Domestica（苹果）MYB10 转录因子基因的上游，当以杂合状态存在时会导致红色果实着色，这是我们证明该方法效率的范例：它包含一个基因组区域，该区域具有通常被短读长测序技术忽略的长结构变异 在使用聚合酶链式反应产品来指定高分子量苹果 DNA 的切割之前，使用聚合酶链反应产品证明了指导 RNA 的切割特异性。随后使用 ONT MinION 流动槽 (R.9.4.1) 制备富集文库并进行测序。使用 Albacore2 (v2.3.4) 和 Guppy (v3.2.4) 进行碱基调用的 7,056 个 ONT 读取中，中位长度分别为 9.78 和 9.89 kb，与参考苹果基因组比对为 85.35% 和 91.38%。在比对读段中，Albacore2 和 Guppy 分别有 2.98% 和 3.04% 的读段深度为 180 × 和 196 × ，并且只有 5 个基因组位点脱靶，读段深度大于 25 × ，这证明了效率CRISPR-Cas9 切割的富集方法和特异性。我们证明该方法可以在植物基因组区域的单倍型水平上分离和解析单核苷酸和结构变异。CRISPR-Cas9 靶点富集和 ONT 测序的结合为精细定位基因座提供了比基因组行走方法更有效的技术。