Multiplex CRISPR/Cas9-mediated genome editing of the FAD2 gene in rice: a model genome editing system for oil palm

Background

Genome editing employing the CRISPR/Cas9 system has been widely used and has become a promising tool for plant gene functional studies and crop improvement. However, most of the applied CRISPR/Cas9 systems targeting one locus using a sgRNA resulted in low genome editing efficiency.

Results

Here, we demonstrate the modification of the FAD2 gene in rice using a multiplex sgRNA-CRISPR/Cas9 genome editing system. To test the system’s efficiency for targeting multiple loci in rice, we designed two sgRNAs based on FAD2 gene sequence of the Oryza sativa Japonica rice. We then inserted the validated sgRNAs into a CRISPR/Cas9 basic vector to construct pYLCRISPRCas9PUbi-H:OsFAD2. The vector was then transformed into protoplast cells isolated from rice leaf tissue via PEG-mediated transfection, and rice calli using biolistic transformation. Direct DNA sequencing of PCR products revealed mutations consisting of deletions of the DNA region between the two target sgRNAs.

Conclusion

The results suggested that the application of the multiplex sgRNA-CRISPR/Cas9 genome editing system may be useful for crop improvement in monocot species that are recalcitrant to genetic modification, such as oil palm.

Loại tài liệu:

Article - Bài báo

Tác giả:

Bahariah, Bohari

Đề mục:

Journal of Genetic Engineering and Biotechnology

Nhà xuất bản:

Elsevier

Ngày xuất bản:

December 2021

Số trang/ tờ:

Định dạng:

pdf

Định danh tư liệu:

DOI: https://doi.org/10.1186/s43141-021-00185-4 | ISSN 1687-157X

Nguồn gốc:

Journal of Genetic Engineering and Biotechnology, Volume 19, Issue 1, December 2021, 86

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