Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA.
Center for Precision Plant Genomics, University of Minnesota, St. Paul, MN 55108, USA.
Int J Mol Sci. 2021 Sep 8;22(18):9723. doi: 10.3390/ijms22189723.
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 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.
高通量分子分析基因靶向 (GT) 事件受到供体分子残留预设的技术挑战。大的供体分子限制引物的放置，导致长的扩增子不能用标准的 NGS 或基于 qPCR 的方法（如 ddPCR）进行分析。在植物中，去除多余的供体既费时又费资源，通常需要植物再生和数周到数月的努力。在这里，我们利用牛津纳米孔扩增子测序（ONAS）来绕过与靶标具有 1 kb 同源性的供体分子的限制，在叶片中的三个基因座上剖析 GT 结果。我们开发了一种新的生物信息学管道，Phased ANalysis of Genome Editing Amplicons（PANGEA），以减少 ONAS 错误对扩增子分析的影响，并捕获了数万种体细胞植物 GT 事件。此外，PANGEA 允许我们在没有选择的情况下在试剂投递后 5 天收集数千个 GT 转化片段，揭示出当整合 18 个或 3 个插入时，大多数事件利用的片段长度小于 100bp。这些数据表明 ONAS 和 PANGEA 对植物 GT 分析有用，并为未来的植物 GT 优化提供了机制基础。
