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Engineering hypercompact IscB nucleases for efficient and versatile genome editing in rice.

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Researchers engineered a novel miniature genome editing system, IscB (Insertion sequences Cas9-like OrfB), for efficient gene editing in rice. This IscB toolkit demonstrates high specificity and versatility, significantly advancing crop breeding applications.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Plant Science

Background:

  • IscB (Insertion sequences Cas9-like OrfB) are novel, small RNA-guided nucleases.
  • Engineered IscB variants show potential for mammalian genome editing.
  • Natural IscB efficiency in eukaryotes is limited, necessitating optimization.

Purpose of the Study:

  • To screen and identify high-activity IscB variants for efficient genome editing in rice.
  • To develop versatile base editors (CBEs and ABEs) from IscB scaffolds.
  • To enhance the utility of IscB systems for crop improvement.

Main Methods:

  • Screening of IscB variants, including enOgeuIscB and ωRNA-v13, for rice genome editing.
  • Development and testing of IscB-based cytosine base editors (CBEs) and adenine base editors (ABEs).
  • Evaluation of deaminase activity (Sdd7 vs. APOBEC3A) and TadA8e fusions for base editing.

Main Results:

  • The pIscB-v3 system achieved an average editing efficiency of 17.61% across ten endogenous rice targets.
  • High editing efficiency (up to 83.33% in T0 generation) with substantial homozygous and bi-allelic mutations was observed.
  • IscB-CBEs utilizing Sdd7 deaminase showed effective C-to-T conversions (up to 47.92%), outperforming human APOBEC3A.
  • Enhanced adenine base editor activity was achieved through TadA8e fusion strategies.

Conclusions:

  • IscB systems offer robust capabilities for developing miniature plant genome editing toolkits.
  • The engineered IscB variants significantly facilitate efficient and versatile crop breeding.
  • This study establishes IscB as a powerful tool for advancing agricultural biotechnology.