Strategies for developing and evaluating CRISPR/CAS9 construct towards drought tolerance in rice (Oryza sativa ssp. Indica)

The majority of the world's population consumes rice (Oryza sativa), which is a staple meal. On the other side, climate change has put rice production in jeopardy. The advent of genome editing technologies has ushered in a new age of agricultural development. Hence, the goal of this work is to...

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Main Authors: Samsulrizal, Nurul Hidayah, Md Yusof, Anis Afuza, Abdul Sattar, Siti Syafiqa, Mohd Zim, Nurul Asyikin, Salleh, Mohd Syahmi, Zainal, Zamri, Zainuddin, Zarina
Format: Conference or Workshop Item
Language:English
Published: 2022
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Online Access:http://irep.iium.edu.my/98515/1/98515_Strategies%20for%20developing%20and%20evaluating%20crisprcas9.pdf
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Institution: Universiti Islam Antarabangsa Malaysia
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spelling my.iium.irep.985152022-07-08T05:11:29Z http://irep.iium.edu.my/98515/ Strategies for developing and evaluating CRISPR/CAS9 construct towards drought tolerance in rice (Oryza sativa ssp. Indica) Samsulrizal, Nurul Hidayah Md Yusof, Anis Afuza Abdul Sattar, Siti Syafiqa Mohd Zim, Nurul Asyikin Salleh, Mohd Syahmi Zainal, Zamri Zainuddin, Zarina S Agriculture (General) S494.5.S86 Sustainable agriculture The majority of the world's population consumes rice (Oryza sativa), which is a staple meal. On the other side, climate change has put rice production in jeopardy. The advent of genome editing technologies has ushered in a new age of agricultural development. Hence, the goal of this work is to use the golden gate cloning approach to produce the CRISPR/Cas9 drought tolerance construct in O. sativa subsp. indica. The development of CRISPR/Cas9 constructs involved several stages, i.e., identification of the SUMO E2-Conjugating Enzyme (OsSCE1) gene, sgRNA selection, and vector construction of the CRISPR/Cas9. The first step in this research was to characterise the OsSCE1 gene using FGENESH, GeneMarkS, InterProScan, and Blast2GO tools. Then, the putative OsSCE1 gene was verified by sequencing, and found to be 99% identical to O. sativa on chromosome 10. The sgRNA design was performed manually with the help of gRNA prediction programmes including WU-CRISPR, CCTop, Benchling, and CRISPR-P. The golden gate cloning method was then used to validate the virtual vector, which included separate vector containing the OsSCE1 sgRNA sequence, planta selectable maker, and Cas9 cassette that were standardised elements for hierarchical modular cloning (MoClo). This study demonstrated that our system, such inclusions are achievable, resulting in a speedier and more efficient assembly process which could contribute to enhancing the resistance and tolerance of rice. To assure a satisfactory outcome, the effectiveness of knocking out candidate genes in O. sativa on drought tolerance will be explored further during the in vivo stage. 2022 Conference or Workshop Item NonPeerReviewed application/pdf en http://irep.iium.edu.my/98515/1/98515_Strategies%20for%20developing%20and%20evaluating%20crisprcas9.pdf Samsulrizal, Nurul Hidayah and Md Yusof, Anis Afuza and Abdul Sattar, Siti Syafiqa and Mohd Zim, Nurul Asyikin and Salleh, Mohd Syahmi and Zainal, Zamri and Zainuddin, Zarina (2022) Strategies for developing and evaluating CRISPR/CAS9 construct towards drought tolerance in rice (Oryza sativa ssp. Indica). In: International Applied Biology Conference 2022, 3-5 June 2022, Melaka, Malaysia. (Unpublished)
institution Universiti Islam Antarabangsa Malaysia
building IIUM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider International Islamic University Malaysia
content_source IIUM Repository (IREP)
url_provider http://irep.iium.edu.my/
language English
topic S Agriculture (General)
S494.5.S86 Sustainable agriculture
spellingShingle S Agriculture (General)
S494.5.S86 Sustainable agriculture
Samsulrizal, Nurul Hidayah
Md Yusof, Anis Afuza
Abdul Sattar, Siti Syafiqa
Mohd Zim, Nurul Asyikin
Salleh, Mohd Syahmi
Zainal, Zamri
Zainuddin, Zarina
Strategies for developing and evaluating CRISPR/CAS9 construct towards drought tolerance in rice (Oryza sativa ssp. Indica)
description The majority of the world's population consumes rice (Oryza sativa), which is a staple meal. On the other side, climate change has put rice production in jeopardy. The advent of genome editing technologies has ushered in a new age of agricultural development. Hence, the goal of this work is to use the golden gate cloning approach to produce the CRISPR/Cas9 drought tolerance construct in O. sativa subsp. indica. The development of CRISPR/Cas9 constructs involved several stages, i.e., identification of the SUMO E2-Conjugating Enzyme (OsSCE1) gene, sgRNA selection, and vector construction of the CRISPR/Cas9. The first step in this research was to characterise the OsSCE1 gene using FGENESH, GeneMarkS, InterProScan, and Blast2GO tools. Then, the putative OsSCE1 gene was verified by sequencing, and found to be 99% identical to O. sativa on chromosome 10. The sgRNA design was performed manually with the help of gRNA prediction programmes including WU-CRISPR, CCTop, Benchling, and CRISPR-P. The golden gate cloning method was then used to validate the virtual vector, which included separate vector containing the OsSCE1 sgRNA sequence, planta selectable maker, and Cas9 cassette that were standardised elements for hierarchical modular cloning (MoClo). This study demonstrated that our system, such inclusions are achievable, resulting in a speedier and more efficient assembly process which could contribute to enhancing the resistance and tolerance of rice. To assure a satisfactory outcome, the effectiveness of knocking out candidate genes in O. sativa on drought tolerance will be explored further during the in vivo stage.
format Conference or Workshop Item
author Samsulrizal, Nurul Hidayah
Md Yusof, Anis Afuza
Abdul Sattar, Siti Syafiqa
Mohd Zim, Nurul Asyikin
Salleh, Mohd Syahmi
Zainal, Zamri
Zainuddin, Zarina
author_facet Samsulrizal, Nurul Hidayah
Md Yusof, Anis Afuza
Abdul Sattar, Siti Syafiqa
Mohd Zim, Nurul Asyikin
Salleh, Mohd Syahmi
Zainal, Zamri
Zainuddin, Zarina
author_sort Samsulrizal, Nurul Hidayah
title Strategies for developing and evaluating CRISPR/CAS9 construct towards drought tolerance in rice (Oryza sativa ssp. Indica)
title_short Strategies for developing and evaluating CRISPR/CAS9 construct towards drought tolerance in rice (Oryza sativa ssp. Indica)
title_full Strategies for developing and evaluating CRISPR/CAS9 construct towards drought tolerance in rice (Oryza sativa ssp. Indica)
title_fullStr Strategies for developing and evaluating CRISPR/CAS9 construct towards drought tolerance in rice (Oryza sativa ssp. Indica)
title_full_unstemmed Strategies for developing and evaluating CRISPR/CAS9 construct towards drought tolerance in rice (Oryza sativa ssp. Indica)
title_sort strategies for developing and evaluating crispr/cas9 construct towards drought tolerance in rice (oryza sativa ssp. indica)
publishDate 2022
url http://irep.iium.edu.my/98515/1/98515_Strategies%20for%20developing%20and%20evaluating%20crisprcas9.pdf
http://irep.iium.edu.my/98515/
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