Creating saponin‐free yellow pea seeds by <i>CRISPR/Cas9</i>‐enabled mutagenesis on β‐amyrin synthase

Hodgins, Connor L.; Salama, Eman M.; Kumar, Rahul; Zhao, Yang; Roth, Susan A.; Cheung, Irene Z.; Chen, Jieyu; Arganosa, Gene C.; Warkentin, Thomas D.; Bhowmik, Pankaj; Ham, Byung‐Kook; Ro, Dae‐Kyun

doi:10.1002/pld3.563

Cited by 6 publications

(1 citation statement)

References 45 publications

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“…This further highlights that we lack sophisticated knowledge of the biophysical affinity of gRNA/Cas9 complex to targets in plant cells. This is in agreement with the well-known lack of predictive power of in silico gRNA activity tools, suggesting that in planta optimization of gRNA choice in protoplasts, hairy roots, or similar systems is a good first step when any off-target mutations are unacceptable (Naim et al, 2020;Hodgins et al, 2024). Still, four out of six gRNAs had no off-target mutagenic potential we could detect.…”

Section: Surprising Divergence Of On-and Offtarget Sequencessupporting

confidence: 83%

Rare but diverse off-target and somatic mutations found in field and greenhouse grown trees expressing CRISPR/Cas9

Goralogia,

Andreatta,

Conrad

et al. 2024

Front. Bioeng. Biotechnol.

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Introduction: CRISPR gene editing, while highly efficient in creating desired mutations, also has the potential to cause off-target mutations. This risk is especially high in clonally propagated plants, where editing reagents may remain in the genome for long periods of time or in perpetuity. We studied a diverse population of Populus and Eucalyptus trees that had CRISPR/Cas9-containing transgenes that targeted one or two types of floral development genes, homologs of LEAFY and AGAMOUS.Methods: Using a targeted sequence approach, we studied approximately 20,000 genomic sites with degenerate sequence homology of up to five base pairs relative to guide RNA (gRNA) target sites. We analyzed those sites in 96 individual tree samples that represented 37 independent insertion events containing one or multiples of six unique gRNAs.Results: We found low rates of off-target mutations, with rates of 1.2 × 10−9 in poplar and 3.1 × 10−10 in eucalypts, respectively, comparable to that expected due to sexual reproduction. The rates of mutation were highly idiosyncratic among sites and not predicted by sequence similarity to the target sites; a subset of two gRNAs showed off-target editing of four unique genomic sites with up to five mismatches relative to the true target sites, reaching fixation in some gene insertion events and clonal ramets. The location of off-target mutations relative to the PAM site were essentially identical to that seen with on-target CRISPR mutations.Discussion: The low rates observed support many other studies in plants that suggest that the rates of off-target mutagenesis from CRISPR/Cas9 transgenes are negligible; our study extends this conclusion to trees and other long-lived plants where CRISPR/Cas9 transgenes were present in the genome for approximately four years.

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Section: Surprising Divergence Of On-and Offtarget Sequencessupporting

confidence: 83%

Rare but diverse off-target and somatic mutations found in field and greenhouse grown trees expressing CRISPR/Cas9

Goralogia,

Andreatta,

Conrad

et al. 2024

Front. Bioeng. Biotechnol.

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A review of plant anti-nutritional factors in animal health and production: The classification, biological properties, and the passivation strategy

Khajali,

Rafiei

2024

Journal of Agriculture and Food Research

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CRISPR/Cas‐Mediated Gene Editing in Plant Immunity and Its Potential for the Future Development of Fungal, Oomycete, and Bacterial Pathogen‐Resistant Pulse Crops

Singer,

Mukthar,

Subedi

et al. 2024

Plant Cell & Environment

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Pulses provide myriad health benefits and are advantageous in an environmental context as a result of their leguminous nature. However, phytopathogenic fungi, oomycetes and bacteria pose a substantial threat to pulse production, at times leading to crop failure. Unfortunately, existing disease management strategies often provide insufficient control, and there is a clear need for the development of new pulse cultivars with durable and broad‐spectrum disease resistance. CRISPR/Cas‐mediated gene editing has proven its potential for rapidly enhancing disease resistance in many plant species. However, this tool has only very recently been applied in pulse species, and never in the context of plant immunity. In this review, we examine the recent successful utilization of this technology in pulse species for proof‐of‐concept or the improvement of other traits. In addition, we consider various genes that have been edited in other plant species to reduce susceptibility to pathogens, and discuss current knowledge regarding their roles in pulses. Given the functional conservation of the selected genes across diverse plant species, there is a high likelihood that their editing would elicit similar effects in non‐oilseed grain legumes, thus providing a suite of potential targets for CRISPR/Cas‐mediated gene editing to promote pulse crop productivity in coming years.

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Creating saponin‐free yellow pea seeds by CRISPR/Cas9‐enabled mutagenesis on β‐amyrin synthase

Cited by 6 publications

References 45 publications

Rare but diverse off-target and somatic mutations found in field and greenhouse grown trees expressing CRISPR/Cas9

Rare but diverse off-target and somatic mutations found in field and greenhouse grown trees expressing CRISPR/Cas9

A review of plant anti-nutritional factors in animal health and production: The classification, biological properties, and the passivation strategy

CRISPR/Cas‐Mediated Gene Editing in Plant Immunity and Its Potential for the Future Development of Fungal, Oomycete, and Bacterial Pathogen‐Resistant Pulse Crops

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