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<p>Plants exhibit genetic redundancy, which requires the mutation of multiple genes for functional analysis. The CRISPR/Cas9 system with polycistronic tRNA-gRNA (PTG) arrays is highly effective for multiplex genes editing in rice. Previously, we introduced a Speed Cloning method for construction of CRISPR/Cas9 vectors with a tandem-arrayed tRNA–gRNA structure using Gibson assembly. Here, we introduce an advanced method, Hyper Cloning, which enhances cloning efficiency beyond that of Speed Cloning by reducing the number of fragments required for assembly. By using the gRNAs in the pRGEB32t vector, which is nonfunctional in the construct generated through the Speed Cloning method, we reduce the number of fragments needed for assembly and increase cloning efficiency. Additionally, we experimentally demonstrated the enhanced performance of the Hyper Cloning method for CRISPR vectors with three, four, and five targets. Ultimately, Hyper Cloning can enhance cloning efficiency of CRISPR/Cas9 vectors with a tandem-arrayed tRNA–gRNA structure.</p>

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Hyper Cloning: A High-Efficiency Method for CRISPR/Cas9 Multiplex Genome Editing in Rice

  • 期刊论文

  • 发表日期：

    2025年6月4日

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  • Min-Jung Kim,
  • Jun-Hyup Kim,
  • Ye-Eun Han,
  • Yunshil Gho,
  • Sunok Moon,
  • Ki-Hong Jung

摘要

Plants exhibit genetic redundancy, which requires the mutation of multiple genes for functional analysis. The CRISPR/Cas9 system with polycistronic tRNA-gRNA (PTG) arrays is highly effective for multiplex genes editing in rice. Previously, we introduced a Speed Cloning method for construction of CRISPR/Cas9 vectors with a tandem-arrayed tRNA–gRNA structure using Gibson assembly. Here, we introduce an advanced method, Hyper Cloning, which enhances cloning efficiency beyond that of Speed Cloning by reducing the number of fragments required for assembly. By using the gRNAs in the pRGEB32t vector, which is nonfunctional in the construct generated through the Speed Cloning method, we reduce the number of fragments needed for assembly and increase cloning efficiency. Additionally, we experimentally demonstrated the enhanced performance of the Hyper Cloning method for CRISPR vectors with three, four, and five targets. Ultimately, Hyper Cloning can enhance cloning efficiency of CRISPR/Cas9 vectors with a tandem-arrayed tRNA–gRNA structure.