Analyzing plant gene targeting outcomes and conversion tracts with nanopore sequencing

Paul A Atkins, Maria Elena S Gamo, Daniel F. Voytas

Research output: Contribution to journalArticlepeer-review

Abstract

The high-throughput molecular analysis of gene targeting (GT) events is made technically challenging by the residual presetabce of donor molecules. Large donor molecules restrict primer placement, resulting in long amplicons that cannot be readily analyzed using standard NGS pipelines or qPCR-based approaches such as ddPCR. In plants, removal of excess donor is time and resource intensive, often requiring plant regeneration and weeks to months of effort. Here, we utilized Oxford Nanopore Amplicon Sequencing (ONAS) to bypass the limitations imposed by donor molecules with 1 kb of homology to the target and dissected GT outcomes at three loci in Nicotiana benthamia leaves. We developed a novel bioinformatic pipeline, Phased ANalysis of Genome Editing Amplicons (PANGEA), to reduce the effect of ONAS error on amplicon analysis and captured tens of thousands of somatic plant GT events. Additionally, PANGEA allowed us to collect thousands of GT conversion tracts 5 days after reagent delivery with no selection, revealing that most events utilized tracts less than 100 bp in length when incorporating an 18 bp or 3 bp insertion. These data demonstrate the usefulness of ONAS and PANGEA for plant GT analysis and provide a mechanistic basis for future plant GT optimization.

Original languageEnglish (US)
Article number9723
JournalInternational journal of molecular sciences
Volume22
Issue number18
DOIs
StatePublished - Sep 8 2021

Bibliographical note

Funding Information:
Funding: This work was supported by a grant from MnDRIVE—a partnership between the University of Minnesota and the State of Minnesota.

Publisher Copyright:
© 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Keywords

  • Amplicon sequencing
  • Bioinformatics
  • Genome engineering
  • Homologous recombination

PubMed: MeSH publication types

  • Journal Article

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