Performing Department
(N/A)
Non Technical Summary
Targeted genetic modifications have the potential to create novel agricultural products that are free of foreign DNA. Modifications that involve a single mutation are exempt from the regulatory process applied to traditional bioengineered crops. However, multiple gene copies or members of a gene family contribute to many agronomically important plant phenotypes. Therefore, a single mutation is insufficient to induce the desired phenotype. For federal regulatory agencies to make science-based decisions, it is necessary to determine if modifications of multiple gene copies or gene family members pose an increased risk to the environment or human health. We will investigate the potential for unintended changes due to CRISPR/Cas9 modification of five agronomically important gene families in soybean where multiple gene copies must be modified to induce a desired phenotype. We will use long-read DNA sequencing to characterize the full genomic complement of the five gene families in two soybean varieties. CRISPR/Cas9 edits generate double-stranded breaks in DNA that can induce chromosomal structural variants. Thus, we will use long-read sequencing to detect structural and nucleotide sequence variants potentially induced by CRISPR/Cas9 edits of multiple linked and dispersed copies of gene family members. Visual comparisons and hyperspectral analysis will be used to identify any unintended phenotypic changes in modified plants. Our data will increase understanding of the environmental risks associated with CRISPR/Cas9-induced changes in an important crop plant and provide a contrast with variation induced by unregulated practices such as conventional breeding and mutagenesis.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
Objective 1.Use long-read DNA sequencing to characterize duplicate genes in two soybean experimental lines.Objective 2.Characterize CRISPR modifications at duplicate genes that vary in linkage, copy number, sequence divergence, and phenotypic effect.Objective 3.Use long-read DNA sequencing and phenotypic screening to identify potential off-target or unintended changes induced by multiple CRISPR edits.
Project Methods
Objective 1. Use long-read DNA sequencing to characterize duplicate genes in two soybean experimental lines. (Years 1 - 2).In Objective 1, we address the fundamental challenge of working with duplicate genes; often, the number of copies of each gene is unknown. We aim to determine the number of complete, partial, and pseudogene copies of each of the genes we target in Bert and Williams82 before making comparisons with CRISPR-modified lines. Of course, sequence similarity can vary among gene copies, so the most important factor is the sequence similarity of gene copies at the genic positions that match guide RNAs (gRNAs) used to target them.Several approaches can be combined for this characterization, including mapping existing short reads to canonical versions of each gene to measure read depth and, thus, copy number. We can also perform local reassembly of existing Pac-Bio reads to improve the characterization of each region. Ultimately, we expect long and accurate Oxford Nanopore Technologies (ONT) sequences to provide a direct read-through of these five genes and their various copies in Bert and Williams82. Thus the product of this objective will be the full-length, sequence-level characterization of the five duplicate genes we are targeting in both Bert and Williams82.Objective 2. Characterize CRISPR modifications at duplicate genes that vary in linkage, copy number, sequence divergence, and phenotypic effect. (Years 1 - 3). In Objective 2, we will assess CRISPR edits produced to create phenotypic changes that improve the agronomic adaptation, nutritional value, and utility of soybean. Each CRISPR-modified soybean line will be grown in the greenhouse and monitored for phenotypic changes resulting from genetic modification. We will assess modified lines for changes related to the modified genes. For the Gibberellin 2-oxidase 8, this will involve changes in plant height or architecture, while the other four gene families affect seed traits that are assessed through proteomic and digestibility studies.Phenotypic characterization of modified lines in this objective will help select lines further characterized by long-read sequencing in Objective 3Objective 3. Use long-read DNA sequencing and phenotypic screening to identify potential off-target or unintended changes induced by multiple CRISPR edits. (Years 1 - 3).We will generate ONTwhole-genome long-read sequencing for ≥ 12 CRISPR-modified lines relative to unmodified versions of the transformation germplasm. The sequence will be used to identify targeted, non-targeted, and unintended changes in soybean genomes. Long sequence reads from individual lines will be mapped to assemblies of 'Bert' and 'Williams82' supplemented with fully characterized versions of the duplicate genes generated under Objective 1.While we will pay special attention to the duplicate genes targeted for CRISPR modification, DNA sequence data is inherently whole-genome, and structural changes anywhere in the genome will be identified. Because modification of multiple CRISPR targets could induce structure rearrangements, will use at least two current approaches that take advantage of ONT sequence for structure variant detection.