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. Result...

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Published inJournal of Genetic Engineering and Biotechnology Vol. 19; no. 1; pp. 86 - 13
Main Authors Bahariah, Bohari, Masani, Mat Yunus Abdul, Rasid, Omar Abd, Parveez, Ghulam Kadir Ahmad
Format Journal Article
LanguageEnglish
Published Berlin/Heidelberg Springer Berlin Heidelberg 11.06.2021
Springer
Springer Nature B.V
Elsevier
Subjects
biolistics
Biomedical Engineering and Bioengineering
biotechnology
callus
CRISPR
CRISPR-Cas systems
Crop improvement
Deoxyribonucleic acid
DNA
DNA sequencing
Editing
Efficiency
Elaeis guineensis
Engineering
FAD2
FAD2 gene
Fatty acids
Gene sequencing
Genes
genetic engineering
Genetic modification
Genetic transformation
Genetically altered foods
Genetically modified organisms
Genome editing
Genomes
Genomics
High oleic acid
Laboratories
leaves
loci
Model monocot
Multiplex CRISPR/Cas9
Multiplexing
Mutagenesis
Mutation
nucleotide sequences
Nucleotide sequencing
Oryza sativa
Plant genetics
protoplasts
Rice
RNA polymerase
Seeds
Transfection
China
United States > US
Model monocot
FAD2
Genome editing
High oleic acid
Multiplex CRISPR/Cas9
Rice
Online AccessGet full text
ISSN1687-157X
2090-5920
DOI10.1186/s43141-021-00185-4

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Abstract 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.
AbstractList 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.
Abstract 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.
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. 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. 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.
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.
BackgroundGenome 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.ResultsHere, 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.ConclusionThe 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.
ArticleNumber 86
Audience Academic
Author Bahariah, Bohari
Rasid, Omar Abd
Masani, Mat Yunus Abdul
Parveez, Ghulam Kadir Ahmad
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  surname: Parveez
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  organization: Advanced Biotechnology and Breeding Centre (ABBC) Division, Malaysian Palm Oil Board (MPOB)
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Issue 1
Keywords Model monocot
FAD2
Genome editing
High oleic acid
Multiplex CRISPR/Cas9
Rice
Language English
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Snippet 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...
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...
BackgroundGenome editing employing the CRISPR/Cas9 system has been widely used and has become a promising tool for plant gene functional studies and crop...
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....
Abstract Background Genome editing employing the CRISPR/Cas9 system has been widely used and has become a promising tool for plant gene functional studies and...
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StartPage 86
SubjectTerms biolistics
Biomedical Engineering and Bioengineering
biotechnology
callus
CRISPR
CRISPR-Cas systems
Crop improvement
Deoxyribonucleic acid
DNA
DNA sequencing
Editing
Efficiency
Elaeis guineensis
Engineering
FAD2
FAD2 gene
Fatty acids
Gene sequencing
Genes
genetic engineering
Genetic modification
Genetic transformation
Genetically altered foods
Genetically modified organisms
Genome editing
Genomes
Genomics
High oleic acid
Laboratories
leaves
loci
Model monocot
Multiplex CRISPR/Cas9
Multiplexing
Mutagenesis
Mutation
nucleotide sequences
Nucleotide sequencing
Oryza sativa
Plant genetics
protoplasts
Rice
RNA polymerase
Seeds
Transfection
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Title Multiplex CRISPR/Cas9-mediated genome editing of the FAD2 gene in rice: a model genome editing system for oil palm
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Volume 19
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