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.
Highlights
The ability to efficiently modify any sequence within a plant genome would greatly accelerate basic and applied plant research
One method for making nucleotide-specific modifications is gene targeting (GT), wherein a locus is repaired by homologous recombination using a supplied ‘donor’ DNA as a template [1]
Primers placed within the homology arms of a donor molecule will amplify both the target locus and donor molecules creating false positives, while primers placed outside the regions of homology will typically yield amplicons too large for analyses
Summary
The ability to efficiently modify any sequence within a plant genome would greatly accelerate basic and applied plant research. Donors with imperfect homology performed poorly even when delivered with a GVR and nuclease, with fold reductions in GT frequency ranging from 0.38 at AGAMOUS and 0.07 at PDS3.2 when delivered donors diverged by 3.33% (regular SNPs every 30 bp) or 10% (natural variation between PDS paralogs), respectively (Figure 2C). It should be noted that the 4th innermost SNP in the PDS natural variant donor is 153bp away from the targeted insertion, making the 3 SNPs inside that range possible background based on SNP patterns in non-GT reads (Figure S12). PANGEA readily accommodates ONAS error when searching for GT events and when examining conversion tracts, making direct and high-throughput mechanistic analysis of GT outcomes within virtually any tissue possible. Our analysis sheds light onto the nature of somatic plant gene targeting events, the high usage of extremely short conversion tracts, and establishes a platform for guiding future GT optimization with mechanistic insights
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