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In silico analysis of OSRR22 isolated from MR 219 rice and the strategy for developing CRISPR/CAS9 construct for genome editing

The rice response regulator 22 (RR22) has been reported to be negatively regulating salt tolerance in Oryza sativa and involved in the cytokinin signaling pathway; however, it has not been documented in any Malaysian rice and is deemed as an appealing subject for CRIPSR/Cas9 editing. This study anal...

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Main Authors: Tamizi, Amin-Asyraf, Samsulrizal, Nurul Hidayah, Sekeli, Rogayah, Zainuddin, Zarina
Format: Conference or Workshop Item
Language:English
English
English
English
Published: 2023
Subjects:
S Agriculture (General)
Online Access:http://irep.iium.edu.my/104104/1/MiGC15-2023-Abstract-AMINASYRAF.pdf
http://irep.iium.edu.my/104104/2/C_MiGC15_Poster%20139-087.pdf
http://irep.iium.edu.my/104104/3/ACCEPTANCE%20LETTER_AMIN%20ASTRAF%20TAMIZI.pdf
http://irep.iium.edu.my/104104/4/MiGC15_Final_Announcement_MiGC15_Brochure.pdf
http://irep.iium.edu.my/104104/
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spelling my.iium.irep.1041042023-03-23T01:39:14Z http://irep.iium.edu.my/104104/ In silico analysis of OSRR22 isolated from MR 219 rice and the strategy for developing CRISPR/CAS9 construct for genome editing Tamizi, Amin-Asyraf Samsulrizal, Nurul Hidayah Sekeli, Rogayah Zainuddin, Zarina S Agriculture (General) The rice response regulator 22 (RR22) has been reported to be negatively regulating salt tolerance in Oryza sativa and involved in the cytokinin signaling pathway; however, it has not been documented in any Malaysian rice and is deemed as an appealing subject for CRIPSR/Cas9 editing. This study analysed the OsRR22 gene from the Malaysian rice cultivar ‘MR 219’ to elucidate its function and determine the gRNA target site, in which the finding served as the most vital part for our current CRISPR/Cas9 genome editing experiment. In brief, the methods employed include total genome isolation, polymerase chain reaction (PCR) amplification, DNA sequencing, genome search, and computational analyses involving an array of in silico tools. The transcript of OsRR22 was 2,019 bp long, composed of six exons and it encoded a highly conserved 696 amino acid residues. Motif analysis revealed the gene product, RR22, contained response regulator (RR) receiver domain (position 27-142), disordered domain (position 154-214), and Myb-like DNA-binding domain (position 214-273). Analysis on the protein-protein interaction (PPI) using STRING revealed RR22 interacted with various proteins including RR24, RR B8A7T0, and a set of HPt domain-containing proteins (B8B4B1, B8AYV8, B8BEM5, B8A9E0 and B8B9H1). Analysis of gene expression profiles via the Rice Expression Database (RED) revealed the OsRR22 (Os06g0183100) gene was highly expressed (FPKM>10) in the leaf and root. In addition, there were 15 genes to be co-expressed (Pearson’s r value > 0.85) with the OsRR22 gene of which high-affinity potassium transporter 9 (HAK9, Os07g0679000) was one of them. Based on the first exon of OsRR22 that encoded a part of the RR receiver domain, a CRISPR-gRNA 20-bp spacer was generated through CCTop. The gRNA spacer was synthesised, annealed, and ligated into the CRSPR/Cas9 pRGEB32 vector. The CRISPR/Cas9 construct—targeting MR 219’s OsRR22 and intended for Agrobacterium-based delivery—was successfully developed. Our study here documents the upstream workflow involved in rice genome editing with an emphasis on the gene characterisation through multiple bioinformatics tools. 2023 Conference or Workshop Item PeerReviewed application/pdf en http://irep.iium.edu.my/104104/1/MiGC15-2023-Abstract-AMINASYRAF.pdf application/pdf en http://irep.iium.edu.my/104104/2/C_MiGC15_Poster%20139-087.pdf application/pdf en http://irep.iium.edu.my/104104/3/ACCEPTANCE%20LETTER_AMIN%20ASTRAF%20TAMIZI.pdf application/pdf en http://irep.iium.edu.my/104104/4/MiGC15_Final_Announcement_MiGC15_Brochure.pdf Tamizi, Amin-Asyraf and Samsulrizal, Nurul Hidayah and Sekeli, Rogayah and Zainuddin, Zarina (2023) In silico analysis of OSRR22 isolated from MR 219 rice and the strategy for developing CRISPR/CAS9 construct for genome editing. In: 15th Malaysia International Genetics Congress 2023, 07-09 March 2023, Bangi Resort Hotel. (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
English
English
English
topic S Agriculture (General)
spellingShingle S Agriculture (General)
Tamizi, Amin-Asyraf
Samsulrizal, Nurul Hidayah
Sekeli, Rogayah
Zainuddin, Zarina
In silico analysis of OSRR22 isolated from MR 219 rice and the strategy for developing CRISPR/CAS9 construct for genome editing
description The rice response regulator 22 (RR22) has been reported to be negatively regulating salt tolerance in Oryza sativa and involved in the cytokinin signaling pathway; however, it has not been documented in any Malaysian rice and is deemed as an appealing subject for CRIPSR/Cas9 editing. This study analysed the OsRR22 gene from the Malaysian rice cultivar ‘MR 219’ to elucidate its function and determine the gRNA target site, in which the finding served as the most vital part for our current CRISPR/Cas9 genome editing experiment. In brief, the methods employed include total genome isolation, polymerase chain reaction (PCR) amplification, DNA sequencing, genome search, and computational analyses involving an array of in silico tools. The transcript of OsRR22 was 2,019 bp long, composed of six exons and it encoded a highly conserved 696 amino acid residues. Motif analysis revealed the gene product, RR22, contained response regulator (RR) receiver domain (position 27-142), disordered domain (position 154-214), and Myb-like DNA-binding domain (position 214-273). Analysis on the protein-protein interaction (PPI) using STRING revealed RR22 interacted with various proteins including RR24, RR B8A7T0, and a set of HPt domain-containing proteins (B8B4B1, B8AYV8, B8BEM5, B8A9E0 and B8B9H1). Analysis of gene expression profiles via the Rice Expression Database (RED) revealed the OsRR22 (Os06g0183100) gene was highly expressed (FPKM>10) in the leaf and root. In addition, there were 15 genes to be co-expressed (Pearson’s r value > 0.85) with the OsRR22 gene of which high-affinity potassium transporter 9 (HAK9, Os07g0679000) was one of them. Based on the first exon of OsRR22 that encoded a part of the RR receiver domain, a CRISPR-gRNA 20-bp spacer was generated through CCTop. The gRNA spacer was synthesised, annealed, and ligated into the CRSPR/Cas9 pRGEB32 vector. The CRISPR/Cas9 construct—targeting MR 219’s OsRR22 and intended for Agrobacterium-based delivery—was successfully developed. Our study here documents the upstream workflow involved in rice genome editing with an emphasis on the gene characterisation through multiple bioinformatics tools.
format Conference or Workshop Item
author Tamizi, Amin-Asyraf
Samsulrizal, Nurul Hidayah
Sekeli, Rogayah
Zainuddin, Zarina
author_facet Tamizi, Amin-Asyraf
Samsulrizal, Nurul Hidayah
Sekeli, Rogayah
Zainuddin, Zarina
author_sort Tamizi, Amin-Asyraf
title In silico analysis of OSRR22 isolated from MR 219 rice and the strategy for developing CRISPR/CAS9 construct for genome editing
title_short In silico analysis of OSRR22 isolated from MR 219 rice and the strategy for developing CRISPR/CAS9 construct for genome editing
title_full In silico analysis of OSRR22 isolated from MR 219 rice and the strategy for developing CRISPR/CAS9 construct for genome editing
title_fullStr In silico analysis of OSRR22 isolated from MR 219 rice and the strategy for developing CRISPR/CAS9 construct for genome editing
title_full_unstemmed In silico analysis of OSRR22 isolated from MR 219 rice and the strategy for developing CRISPR/CAS9 construct for genome editing
title_sort in silico analysis of osrr22 isolated from mr 219 rice and the strategy for developing crispr/cas9 construct for genome editing
publishDate 2023
url http://irep.iium.edu.my/104104/1/MiGC15-2023-Abstract-AMINASYRAF.pdf
http://irep.iium.edu.my/104104/2/C_MiGC15_Poster%20139-087.pdf
http://irep.iium.edu.my/104104/3/ACCEPTANCE%20LETTER_AMIN%20ASTRAF%20TAMIZI.pdf
http://irep.iium.edu.my/104104/4/MiGC15_Final_Announcement_MiGC15_Brochure.pdf
http://irep.iium.edu.my/104104/
_version_ 1761616150442016768
score 13.211869

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