Received: 06-08-2022
Accepted: 27-09-2022
DOI:
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Designing CRISPR/CAS9 System for Knocking out OsNRAMP5Related to Cadmium Accumulation in Rice Variety TBR225
,
Nguyen Anh Minh
2
,
Pham Thu Hang
2
,
Pham Phuong Ngoc
2
,
Le Quynh Mai
3
,
Pham Xuan Hoi
2
,
Nguyen Duy Phuong
2
Keywords
Cadmium, CRISPR/Cas9, Knocking out gene, OsNRAMP5, TBR225
Abstract
Cadmium (Cd) is highly toxic to most living organisms, including humans. Rice with excessive Cd (> 0,2 mg/kg - QCVN 8-2:2011/BYT) is one of the leading causes of Cd poisoning, threatening human health. Several genes belonging to the NRAMP(natural resistance-associated macrophage protein) family are related to metal accumulation in plants; in which OsNRAMP5has been identified to be involved in the transport of various metals such as iron (Fe), zinc (Zn), copper (Cu), manganese) Mn and Cd in rice. In order to identify the function of OsNRAMP5in elite rice variety TBR225, we evaluated the expression of the targeted gene and designed Clustered regular interspaced short palindromic repeats/CRISPR-associated Cas9 (CRISPR/Cas9) system for editing TBR225OsNRAMP5. The increase of OsNRAMP5expression was observed in the root tissues of TBR225 rice plant in the Cd-treatment experiment using hydroponic culture. A sequence containing the ExonI region of OsNRAMP5with a size of 0.29kb was isolated from the total DNA of TBR225 to design single guide RNAs (sgRNA) for OsNRAMP5-editingCRISPR/Cas9 system. The sgRNA expression construct carrying two CRISPR RNA (crRNA) sequences recognizing two different sites on the ExonI of OsNRAMP5was ligated into the binary vector pCas9. The recombinant vector was validatedby PCR and sequencinganalysis. The results serve as basis for the improvement ofTBR225 rice variety with high quality byusinggenome editing technology.
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