CRISPR-Cas9 enrichment and long read sequencing for fine mapping in plants
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nt Methods Open Access
METHODOLOGY
CRISPR‑Cas9 enrichment and long read sequencing for fine mapping in plants Elena López‑Girona1* , Marcus W. Davy2 , Nick W. Albert1 , Elena Hilario3 , Maia E. M. Smart1 , Chris Kirk1 , Susan J. Thomson4 and David Chagné1
Abstract Background: Genomic methods for identifying causative variants for trait loci applicable to a wide range of germ‑ plasm 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 Nano‑ pore 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 exem‑ plar 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 Alba‑ core2 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 sequenc‑ ing provides a more efficient technology for fine-mapping loci than genome-walking approaches. Keywords: Causative variant, SNP, Apple, MYB10, Red flesh, Oxford nanopore, QTL cloning Background Representatives of most plant species important to primary industries have been fully sequenced following the dramatic reduction of sequencing cost. However, the individuals from which reference genomes have been assembled are often chosen because they are highly *Correspondence: elena.lopez‑[email protected] 1 The New Zealand Institute for Plant and Food Research Limited (Plant & Food Research), Private Bag 11600, Palmerston North 4442, New Zealand Full list of author information is available at the end of the article
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