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Cas9-assisted targeting of DNA fragments in complex genomes is viewed as an essential strategy to obtain high-quality and continuous sequence data. However, the purity of target loci selected by pulsed-field gel electrophoresis (PFGE) has so far been insufficient to assemble the sequence in one contig. Here, we describe the μLAS technology to capture and purify high molecular weight DNA. First, the technology is optimized to perform high sensitivity DNA profiling with a limit of detection of 20 fg/μl for 50 kb fragments and an analytical time of 50 min. Then, μLAS is operated to isolate a 31.5 kb locus cleaved by Cas9 in the genome of the plant Medicago truncatula. Target purification is validated on a Bacterial Artificial Chromosome plasmid, and subsequently carried out in whole genome with μLAS, PFGE or by combining these techniques. PacBio sequencing shows an enrichment factor of the target sequence of 84 with PFGE alone versus 892 by association of PFGE with μLAS. These performances allow us to sequence and assemble one contig of 29 441 bp with 99% sequence identity to the reference sequence.

μLAS technology for DNA isolation coupled to Cas9-assisted targeting for sequencing and assembly of a 30 kb region in plant genome