Design and construction of single guide RNA for CRISPR/Cas9 system based on the xa13 resistance gene in some varieties of rice (Oryza sativa)

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Wulan Arum Hardiyani
Ali Wafa
Wahyu Indra Duwi Fanata
Hardian Susilo Addy


The xa13 gene is a recessive resistance gene against Xanthomonas oryzae pv. oryzae (Xoo) found in several rice varieties. Activation of this gene will trigger the formation of sucrose as a nutrient supply to Xoo for their growth in the plant. The disruption of this recessive gene expression in the plant can affect the negative impact of the gene, and recently can be created using clustered regularly interspaced short palindromic repeats (CRISPR) system using CRISPR-associated protein-9 (CRISPR/Cas9) technology that requires gRNA to recognize the targeted-sequence. This study aimed to design and construct the gRNA-targeting xa13 gene in rice using bioinformatics tools. CHOPCHOP was used for generated the gRNA candidates according to the target gene sequence. Two candidates of gRNA-targeted xa13 have been selected based on the analysis of bioinformatics data. Each candidate of gRNA consisted of 20 nucleotides (nt) of the target sequence upstream 3 nt of the protospacer adjacent motif (PAM) sequence (5’-NGG) targeting two exons in the xa13 gene. The gRNA1 will target exon 1 and the gRNA2 will target exon 2, with an efficiency of 52.51% and 44.63% respectively. Data showed that the GC content of all gRNA candidates ranged from 55–70% with no target-off location in the whole genome of rice. The transformation and confirmation test based on the physiological and genomic characteristics of transformants confirmed that the design has been successfully constructed.

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Hardiyani, W. A.; Wafa, A.; Fanata, W. I. D.; Addy, H. S. Design and Construction of Single Guide RNA for CRISPR/Cas9 System Based on the xa13 Resistance Gene in Some Varieties of Rice (Oryza Sativa). J Trop Plant Pests Dis 2023, 23, 47-55.



Ali Z, Abulfaraj A, Idris A, Ali S, Tashkandi M, & Mahfouz MM. 2015. CRISPR/Cas9-mediated viral interference in plants. Genome Biol. 16: 238.

Bhagwat AM, Graumann J, Wiegandt R, Bentsen M, Welker J, Kuenne C, Preussner J, Braun T, & Looso M. 2020. Multicrispr: gRNA design for prime editing and parallel targeting of thousands of targets. Life Science Alliance. 3(11): e202000757.

Breia R, Conde A, Badim H, Fortes AM, Gerós H, & Granell A. 2021. Plant SWEETs: from sugar transport to plant–pathogen interaction and more unexpected physiological roles. Plant Physiol. 186(2): 836–852.

Cebrián J, Kadomatsu-Hermosa MJ, Castán A, Martínez V, Parra C, Fernández-Nestosa MJ, Schaerer C, Martínez-Robles ML, Hernández P, Krimer DB, Stasiak A, & Schvartzman JB. 2015. Electrophoretic mobility of supercoiled, catenated and knotted DNA molecules. Nucleic Acids Res. 43(4): e24.

Chen S, Wang C, Yang J, Chen B, Wang W, Su J, Feng A, Zeng L, & Zhu X. 2020. Identification of the novel bacterial blight resistance gene Xa46(t) by mapping and expression analysis of the rice mutant H120. Sci. Rep. 10: 12642.

Chu Z, Yuan M, Yao J, Ge X, Yuan B, Xu C, Li X, Fu B, Li Z, Bennetzen JL, Zhang Q, & Wang S. 2006. Promoter mutations of an essential gene for pollen development result in disease resistance in rice. Genes Dev. 20(10): 1250–1255.

Cranenburgh RM, Hanak JAJ, Williams SG, & Sherratt DJ. 2001. Escherichia coli strains that allow antibiotic-free plasmid selection and maintenance by repressor titration. Nucleic Acids Res. 29(5): e26.

Crooks GE, Hon G, Chandonia JM, & Brenner SE. 2004. WebLogo: a sequence logo generator. Genome Res. 14(6): 1188–1190.

Donovan S, Mao Y, Orr DJ, Carmo-Silva E, & McCormick AJ. 2020. CRISPR-Cas9-mediated mutagenesis of the rubisco small subunit family in Nicotiana tabacum. Front. Genome Ed. 2: 605614.

Fischer MD, Mgboji E, & Liu Z. 2018. Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes. Plant Methods. 14: 91.

Gagnon JA, Valen E, Thyme SB, Huang P, Ahkmetova L, Pauli A, Montague TG, Zimmerman S, Richter C, & Schier AF. 2014. Efficient mutagenesis byCas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs. PLoS ONE. 9(5): e98186.

Gaj T, Gersbach CA, & Barbas III CF. 2013. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31(7): 397–405.

Hintermann G, Fischer HM, Crameri R, & Hütter R. 1981. Simple procedure for distinguishing CCC, OC, and L forms of plasmid DNA by agarose gel electrophoresis. Plasmid. 5(3): 371–373.

Jiang N, Yan J, Liang Y, Shi Y, He Z, Wu Y, Zeng Q, Liu X, & Peng J. 2020. Resistance genes and their interactions with bacterial blight/leaf streak pathogens (Xanthomonas oryzae) in rice (Oryza sativa L.)-an updated review. Rice. 13: 3.

Khaeruni A, Taufik M, Wijayanto T, & Johan EA. 2014. Perkembangan penyakit hawar daun bakteri pada tiga varietas padi sawah yang diinokulasi pada beberapa fase pertumbuhan [Development of bacterial leaf blight disease inoculated on three varieties of paddy rice at various growth stage]. Jurnal Fitopatologi Indonesia. 10(4): 119–125.

Kim YA, Moon H, & Park CJ. 2019. CRISPR/Cas9-targeted mutagenesis of Os8N3 in rice to confer resistance to Xanthomonas oryzae pv. oryzae. Rice. 12: 67.

Kostylev M, Otwell AE, Richardson RE, & Suzuki Y. 2015. Cloning should be simple: Escherichia coli DH5?-mediated assembly of multiple DNA fragments with short end homologies. PLoS ONE. 10(9): e0137466.

Kumar A, Kumar R, Sengupta D, Das SN, Pandey MK, Bohra A, Sharma NK, Sinha P, Sk H, Ghazi IA, Laha GS, & Sundaram RM. 2020. Deployment of genetic and genomic tools toward gaining a better understanding of rice-Xanthomonas oryzae pv. oryzae interactions for development of durable bacterial blight resistant rice. Front. Plant Sci. 11: 1152.

Labun K, Krause M, Cleuren YT, & Valen E. 2021. CRISPR genome editing made easy through the CHOPCHOP website. Curr. Protoc. 1(4): e46.

Labun K, Montague TG, Gagnon JA, Thyme SB, & Valen, E. 2016. CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Res. 44(W1): W272–W276.

Lee K, Zhu H, Yang B, and Wang K. 2019. An Agrobacterium-mediated CRISPR/Cas9 platform for genome editing in maize. In: Qi Y. (Ed). Plant Genome Editing with CRISPR Systems. Methods in Molecular Biology. Vol 1917. pp. 121–143. Humana Press, New York.

Liakopoulos A, van der Goot J, Bossers A, Betts J, Brouwer MSM, Kant A, Smith H, Ceccarelli D, & Mevius D. 2018. Genomic and functional characterization of IncX3 plasmids encoding blaSHV-12 in Escherichia coli from human and animal origin. Sci. Rep. 8: 7674.

Liu G, Zhang Y, & Zhang T. 2020. Computational approaches for effective CRISPR guide RNA design and evaluation. Comput. Struc. Biotechnol. J. 18: 35–44.

Marillonnet S & Grützner R. 2020. Synthetic DNA assembly using golden gate cloning and the hierarchical modular cloning pipeline. Curr. Protoc. Mol. Biol. 130(1): e115.

Montague TG, Cruz JM, Gagnon JA, Church GM, & Valen E. 2014. CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 42(W1): W401–W407.

Nadhira NE, Wafa A, Fanata WID, & Addy HS. 2022. Resistance gene expression in selected Indonesian pigmented rice varieties against infection by Xanthomonas oryzae pv. oryzae. Indones. J. Biotechnol. 27(2): 51–57.

Nitiss JL, Soans E, Rogojina A, Seth A, & Mishina M. 2012. Topoisomerase assays. Current Protocols in Pharmacology. 57(Suppl 3.3): 1–27.

Pinem T & Syarif Z. 2018. Intensitas serangan Xanthomonas oryzae pv. oryzae pada beberapa varietas padi sawah dan dampaknya terhadap pertumbuhan dan hasil panen [Attack intensity of Xanthomonas oryzae pv. oryzae on several rice varieties and the impact to growth and yield]. Jurnal Proteksi Tanaman (Journal of Plant Protection). 2(1): 9–17.

Potapov V, Ong JL, Kucera RB, Langhorst BW, Bilotti K, Pryor JM, Cantor EJ, Canton B, Knight TF, Evans Jr TC, & Lohman GJS. 2018. Comprehensive profiling of four base overhang ligation fidelity by T4 DNA ligase and application to DNA assembly. ACS Synth. Biol. 7(11): 2665–2674.

Xie K, Minkenberg B, & Yang Y. 2015. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA processing system. PNAS. 112(11): 3570–3575.

Xie K & Yang Y. 2013. RNA-Guided genome editing in plants using a CRISPR-Cas system. Mol. Plant. 6(6): 1975–1983.

Xie K, Zhang J, & Yang Y. 2014. Genome-wide prediction of highly specific guide RNA spacers for CRISPR-Cas9-mediated genome editing in model plants and major crops. Mol. Plant. 7(5): 923–926.

Xu Z, Xu X, Gong Q, Li Z, Li Y, Wang S, Yang Y, Ma W, Liu L, Zhu B, Zou L, & Chen G. 2019. Engineering broad-spectrum bacterial blight resistance by simultaneously disrupting variable TALE-binding elements of multiple susceptibility genes in rice. Mol. Plant. 12(11): 1434–1446.

Yu P, Wang XM, Yuan XP, Wang CH, Xu Q, Feng Y, Yu HY, Wang YP, & Wei XH. 2016. Sequence variations and haplotypes of the bacterial blight resistance gene xa13 in rice. J. Plant Pathol. 98(1): 167–169.

Zafar K, Khan MZ, Amin I, Mukhtar Z, Yasmin S, Arif M, Ejaz K, & Mansoor S. 2020. Precise CRISPR-Cas9 mediated genome editing in super basmati rice for resistance against bacterial blight by targeting the major susceptibility gene. Front. Plant Sci. 11: 575.

Zhang JH, Adikaram P, Pandey M, Genis A, & Simonds WF. 2016. Optimization of genome editing through CRISPR-Cas9 engineering. Bioengineered. 7(3): 166–174.

Zhang K, Yin X, Shi K, Zhang S, Wang J, Zhao S, Deng H, Zhang C, Wu Z, Li Y, Zhou X & Deng W. 2021. A high-efficiency method for site-directed mutagenesis of large plasmids based on large DNA fragment amplification and recombinational ligation. Sci. Rep. 11: 10454.

Zhang Q, Maroof MAS, Lu TY, & Shen BZ. 1992. Genetic diversity and differentiation of indica and japonica rice detected by RFLP analysis. Theoret. Appl. Genetics. 83(4): 495–499.

Zhu H & Liang C. 2019. CRISPR-DT: designing gRNAs for the CRISPR-Cpf1 system with improved target efficiency and specificity. Bioinformatics. 35(16): 2783–2789.