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<p>Genome editing employing CRISPR/Cas9 systems has found widespread applications for knocking out targeted genes. In spite of exponential applications in plants for trait improvement, low editing efficiency in plants is a major concern. We report construction of a pCAMBIA2300 based binary vector cassette (pCR) harbouring novel recombinant CRISPR/Cas9 system for efficient genome editing in plants. The <i>Cas9</i> cDNA with sequence encoding nuclear localization signals at the N-terminal and C-terminal ends had been codon optimized for better expression in plants. Undesirable internal restriction sites were removed. Small stretch of 5’ UTR sequence of Rubisco small subunit (<i>rcbS</i>) of potato, harbouring in between potato granule bound starch synthase (GBSS) intron, was added at the 5’ end of the Cas9 cDNA to function as 5’ UTR. The recombinant Cas9 gene (<i>rdCas9</i>) was placed under the transcriptional control of CaMV 35S promoter and NOS terminator. The single guide RNA cassette (sgRNA) was comprised of <i>Arabidopsis</i> U6 promoter, 20–21 nucleotide (nt) spacer sequence, sgRNA scaffold sequence and potato U6 RNA Pol-III termination sequence. The 20–21 nt sgRNA spacer sequence could be added to the sgRNA construct by <i>Aar</i>I or <i>Paq</i>CI digestion. The sgRNA construct had been designed in such a way so that single or multiplexed sgRNA could be cloned into the pCR vector cassette in a single step. Moreover, modular nature of this vector system can help to derive different combination of promoter, terminator with Cas9 and sgRNA constructs. The efficacy of the pCR vector system had been validated in <i>Nicotiana tabacum</i> and <i>Solanum tuberosum</i> by knocking out phytoene desaturase gene (<i>PDS</i>), through <i>Agrobacterium</i>-mediated transformation. The pCR binary vector system can be utilized as a versatile tool box for efficient genome editing of plant to improve agriculturally important traits.</p>

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A novel recombinant CRISPR/Cas9 vector system for genome editing in plants

  • 期刊论文

  • 发表日期：

    2025年10月8日

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  • Krishnayan Paul,
  • Raman K. Venkat,
  • Mahi Baaniya,
  • Ishwar Jadhav,
  • Sougata Bhattacharjee,
  • Jyotsana Tilgam,
  • Manjesh Saakre,
  • Priyanka Kumari,
  • Suparna Das,
  • Joshitha Vijayan,
  • Rohini Sreevathsa,
  • Debasis Pattanayak

摘要

Genome editing employing CRISPR/Cas9 systems has found widespread applications for knocking out targeted genes. In spite of exponential applications in plants for trait improvement, low editing efficiency in plants is a major concern. We report construction of a pCAMBIA2300 based binary vector cassette (pCR) harbouring novel recombinant CRISPR/Cas9 system for efficient genome editing in plants. The Cas9 cDNA with sequence encoding nuclear localization signals at the N-terminal and C-terminal ends had been codon optimized for better expression in plants. Undesirable internal restriction sites were removed. Small stretch of 5’ UTR sequence of Rubisco small subunit (rcbS) of potato, harbouring in between potato granule bound starch synthase (GBSS) intron, was added at the 5’ end of the Cas9 cDNA to function as 5’ UTR. The recombinant Cas9 gene (rdCas9) was placed under the transcriptional control of CaMV 35S promoter and NOS terminator. The single guide RNA cassette (sgRNA) was comprised of Arabidopsis U6 promoter, 20–21 nucleotide (nt) spacer sequence, sgRNA scaffold sequence and potato U6 RNA Pol-III termination sequence. The 20–21 nt sgRNA spacer sequence could be added to the sgRNA construct by AarI or PaqCI digestion. The sgRNA construct had been designed in such a way so that single or multiplexed sgRNA could be cloned into the pCR vector cassette in a single step. Moreover, modular nature of this vector system can help to derive different combination of promoter, terminator with Cas9 and sgRNA constructs. The efficacy of the pCR vector system had been validated in Nicotiana tabacum and Solanum tuberosum by knocking out phytoene desaturase gene (PDS), through Agrobacterium-mediated transformation. The pCR binary vector system can be utilized as a versatile tool box for efficient genome editing of plant to improve agriculturally important traits.