CRISPR-Cas9 enrichment and long read sequencing for fine mapping in plants
CRISPR-Cas9 enrichment and long read sequencing for fine mapping in plants
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DOI：
10.1186/s13007-020-00661-x
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发表时间：
2020-09-01
期刊：
影响因子：
5.1
通讯作者：
Chagne, David
中科院分区：
文献类型：
--
作者：
Lopez-Girona, Elena;Davy, Marcus W.;Chagne, David
背景植物生物学家和育种者需要一种基因组方法来识别适用于广泛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。结果我们实现了针对苹果的Cas9靶向测序，这是一种结合CRISPR-Cas9靶向切割感兴趣区域的方法，然后使用牛津纳米孔技术(ONT)进行浓缩和长读测序。我们证明了这种方法有能力专门切割和丰富一个跨越8kb的植物基因组座位。位于苹果MYB10转录因子基因上游的重复微卫星基序，在杂合状态下导致红色果实着色，是我们展示这种方法效率的范例：它包含一个基因组区域，其中包含一个通常被短读测序技术忽略的长结构变异。随后利用ONT minion flow cell(R.9.4.1)制备了一个丰富的文库并进行了测序。在使用Albacore2(v2.3.4)和Guppy(v3.2.4)进行碱基调用的7,056个ont中，中位长度分别为9.78和9.89 kb，85.35和91.38%与参考苹果基因组一致。其中Albacore2和Guppy分别为2.98%和3.04%，读取深度分别为180x和196x，只有5个基因组座位的读取深度大于25x，证明了该方法的有效性和CRISPR-Cas9裂解的特异性。结论该方法可以在植物基因组单倍型水平分离和分辨单核苷酸和结构变异。CRISPR-Cas9靶标浓缩和ONT测序的结合为精细定位基因座提供了一种比基因组行走方法更有效的技术。
BackgroundGenomic 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.ResultsWe 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 Malusxdomestica (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 technologiesCleavage 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 180xand 196xfor Albacore2 and Guppy, respectively, and only five genomic loci were off-target with read depth greater than 25x, which demonstrated the efficiency of the enrichment method and specificity of the CRISPR-Cas9 cleavage.ConclusionsWe 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.