Easy detection of CRISPR/Cas9-Induced Insertions, Deletions, and Substitutions in Rice Genes OsMADS26, OsRAC1 and OsNRT1.1b using SYBR Green qPCR and Robust HRM analysis in R software
Abstract
Rice is a major cereal crop for global food and nutritional security and a key target for genetic improvement. CRISPR/Cas9 enables precise genetic modification in crops, but mutation screening remains a technical and economic barrier to broader genome-editing applications.
Although several detection methods are available, some require labor-intensive procedures, specialized equipment, high costs, or limited sensitivity to specific mutation types. Highresolution melting (HRM) analysis is an established approach for screening CRISPR/Casinduced mutations in plants, including rice. Here, we evaluated an adapted HRM workflow combining conventional SYBR Green-based qPCR chemistry with downstream computational analysis to detect CRISPR/Cas9-induced mutations at three rice loci: OsMADS26, OsRAC1, and OsNRT1.1b. The workflow detected insertions, deletions, and base substitutions. Across the three loci, the 1% edited-DNA mixtures showed a slight observable deviation from the wildtype melting profile under the conditions evaluated, although this should not be interpreted as a validated detection threshold. Although the assessment of heterozygous samples was limited by their availability, the results support the potential applicability of the approach for individual sample analysis and expanded sample screening. A customizable R script complemented visual analysis by evaluating melting temperature (Tm) and GCP-derived dissimilarity, supporting sample classification. By combining standard SYBR Green chemistry with an adaptable analysis workflow, the method offers an alternative to dedicated HRM reagents and proprietary platforms. This approach provides a practical and potentially lower-cost option for mutation screening at the evaluated rice loci and may be adapted and validated for other targets and plant species.
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BibTeX
@article{reis2026easy,
title = {{Easy detection of CRISPR/Cas9-Induced Insertions, Deletions, and Substitutions in Rice Genes OsMADS26, OsRAC1 and OsNRT1.1b using SYBR Green qPCR and Robust HRM analysis in R software}},
author = {Bianca Cristina Carvalho Reis and Céline Georget and Anne-Cécile Meunier and Edson Mario de Andrade Silva and Christophe Perin and Léo Herbert and Natasha dos Santos Lopes and Fabienne Micheli and Tiago Antônio de Oliveira Mendes},
journal = {New Biotechnology},
year = {2026},
month = aug,
doi = {10.1016/j.nbt.2026.07.005},
url = {https://hal.inrae.fr/hal-05721448v2},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Easy detection of CRISPR/Cas9-Induced Insertions, Deletions, and Substitutions in Rice Genes OsMADS26, OsRAC1 and OsNRT1.1b using SYBR Green qPCR and Robust HRM analysis in R software
AU - Bianca Cristina Carvalho Reis
AU - Céline Georget
AU - Anne-Cécile Meunier
AU - Edson Mario de Andrade Silva
AU - Christophe Perin
AU - Léo Herbert
AU - Natasha dos Santos Lopes
AU - Fabienne Micheli
AU - Tiago Antônio de Oliveira Mendes
PY - 2026
DA - 2026/08/11
JO - New Biotechnology
DO - 10.1016/j.nbt.2026.07.005
UR - https://hal.inrae.fr/hal-05721448v2
AB - Rice is a major cereal crop for global food and nutritional security and a key target for genetic improvement. CRISPR/Cas9 enables precise genetic modification in crops, but mutation screening remains a technical and economic barrier to broader genome-editing applications. Although several detection methods are available, some require labor-intensive procedures, specialized equipment, high costs, or limited sensitivity to specific mutation types. Highresolution melting (HRM) analysis is an established approach for screening CRISPR/Casinduced mutations in plants, including rice. Here, we evaluated an adapted HRM workflow combining conventional SYBR Green-based qPCR chemistry with downstream computational analysis to detect CRISPR/Cas9-induced mutations at three rice loci: OsMADS26, OsRAC1, and OsNRT1.1b. The workflow detected insertions, deletions, and base substitutions. Across the three loci, the 1% edited-DNA mixtures showed a slight observable deviation from the wildtype melting profile under the conditions evaluated, although this should not be interpreted as a validated detection threshold. Although the assessment of heterozygous samples was limited by their availability, the results support the potential applicability of the approach for individual sample analysis and expanded sample screening. A customizable R script complemented visual analysis by evaluating melting temperature (Tm) and GCP-derived dissimilarity, supporting sample classification. By combining standard SYBR Green chemistry with an adaptable analysis workflow, the method offers an alternative to dedicated HRM reagents and proprietary platforms. This approach provides a practical and potentially lower-cost option for mutation screening at the evaluated rice loci and may be adapted and validated for other targets and plant species.
SN - 1871-6784
SN - 1876-4347
ER -