Long-read sequencing for fast and robust identification of correct genome-edited alleles: PCR-based and Cas9 capture methods.

Autor: Christopher V McCabe, Peter D Price, Gemma F Codner, Alasdair J Allan, Adam Caulder, Skevoulla Christou, Jorik Loeffler, Matthew Mackenzie, Elke Malzer, Joffrey Mianné, Krystian J Nowicki, Edward J O'Neill, Fran J Pike, Marie Hutchison, Benoit Petit-Demoulière, Michelle E Stewart, Hilary Gates, Sara Wells, Nicholas D Sanderson, Lydia Teboul
Jazyk: angličtina
Rok vydání: 2024
Předmět:
Zdroj: PLoS Genetics, Vol 20, Iss 3, p e1011187 (2024)
Druh dokumentu: article
ISSN: 1553-7390
1553-7404
DOI: 10.1371/journal.pgen.1011187&type=printable
Popis: BackgroundRecent developments in CRISPR/Cas9 genome-editing tools have facilitated the introduction of precise alleles, including genetic intervals spanning several kilobases, directly into the embryo. However, the introduction of donor templates, via homology directed repair, can be erroneous or incomplete and these techniques often produce mosaic founder animals. Thus, newly generated alleles must be verified at the sequence level across the targeted locus. Screening for the presence of the desired mutant allele using traditional sequencing methods can be challenging due to the size of the interval to be sequenced, together with the mosaic nature of founders.Methodology/principal findingsIn order to help disentangle the genetic complexity of these animals, we tested the application of Oxford Nanopore Technologies long-read sequencing at the targeted locus and found that the achievable depth of sequencing is sufficient to offset the sequencing error rate associated with the technology used to validate targeted regions of interest. We have assembled an analysis workflow that facilitates interrogating the entire length of a targeted segment in a single read, to confirm that the intended mutant sequence is present in both heterozygous animals and mosaic founders. We used this workflow to compare the output of PCR-based and Cas9 capture-based targeted sequencing for validation of edited alleles.ConclusionTargeted long-read sequencing supports in-depth characterisation of all experimental models that aim to produce knock-in or conditional alleles, including those that contain a mix of genome-edited alleles. PCR- or Cas9 capture-based modalities bring different advantages to the analysis.
Databáze: Directory of Open Access Journals
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