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A circularly permuted CasRx platform for efficient, site-specific RNA editing.

Yuanming Wang1,2, Kaiwen Ivy Liu1,2, Mengying Mandy Liu1,2

  • 1School of Chemistry, Chemical Engineering and Biotechnology, Nanyang Technological University, Singapore, Singapore.

Nature Biotechnology
|October 9, 2024
PubMed
Summary
This summary is machine-generated.

We developed xPERT, an optimized RNA editing platform, by engineering CasRx. xPERT offers high on-target RNA editing and low off-target effects, enabling precise RNA sequence modification without genomic damage.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • RNA Therapeutics

Background:

  • Existing RNA editing tools face challenges with efficacy and specificity.
  • Off-target RNA editing remains a significant hurdle in developing reliable tools.

Purpose of the Study:

  • To develop an optimized RNA editing platform with improved efficacy and specificity.
  • To engineer a CasRx-based tool for programmable RNA editing.

Main Methods:

  • Rational protein engineering of an inactive Cas13 ortholog fused to a mutant ADAR2 deaminase domain.
  • Circular permutation of a CasRx K940L mutant to create a robust scaffold.
  • Benchmarking the developed tool (xPERT) against the REPAIR system.

Main Results:

  • The engineered xPERT platform demonstrates strong on-target RNA editing activity.
  • xPERT exhibits significantly reduced off-target editing compared to previous systems.
  • Topological rearrangement via circular permutation created a robust tethering scaffold for the deaminase domain.

Conclusions:

  • The xPERT platform provides a robust solution for programmable RNA editing.
  • This technology allows for precise alteration of RNA sequences without genomic alteration.
  • xPERT enables temporary cellular modifications and customized protein functions.