Analyzing Plant Gene Targeting Outcomes and Conversion Tracts with Nanopore Sequencing

Area of Science:

• Molecular Biology
• Genetics
• Bioinformatics

Background:

• High-throughput analysis of gene targeting (GT) is hindered by residual donor DNA, complicating amplicon sequencing.
• Standard next-generation sequencing (NGS) and digital droplet PCR (ddPCR) struggle with long amplicons caused by donor interference.
• Donor DNA removal in plants is laborious, requiring extensive time and resources like plant regeneration.

Purpose of the Study:

• To develop and validate a method for high-throughput analysis of plant gene targeting events.
• To overcome technical limitations imposed by donor molecules in amplicon sequencing.
• To characterize gene targeting outcomes and repair tract usage in *Nicotiana benthamiana*.

Main Methods:

• Utilized Oxford Nanopore Amplicon Sequencing (ONAS) to analyze gene targeting events in *Nicotiana benthamiana* leaves.
• Developed a novel bioinformatics pipeline, Phased ANalysis of Genome Editing Amplicons (PANGEA), to process ONAS data and mitigate sequencing errors.
• Applied ONAS and PANGEA to analyze GT outcomes at three distinct genetic loci.

Main Results:

• Successfully captured tens of thousands of somatic plant gene targeting events.
• Characterized thousands of gene targeting conversion tracts within five days of reagent delivery, without selection.
• Demonstrated that most gene targeting events utilized repair tracts shorter than 100 bp for insertions of 18 bp or 3 bp.

Conclusions:

• ONAS coupled with the PANGEA pipeline offers a powerful and efficient approach for plant gene targeting analysis.
• The findings provide mechanistic insights into plant gene targeting repair pathways.
• This methodology facilitates future optimization of gene targeting strategies in plants.