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Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells
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Rewriting endogenous human transcripts with dual CRISPR-guided 3' trans-splicing.

Sita S Chandrasekaran1, Cyrus Tau2, Becky Xu Hua Fu3

  • 1Arc Institute, 3181 Porter Drive, Palo Alto, CA 94304, USA; Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA; University of California, Berkeley, San Francisco Graduate Program in Bioengineering, Berkeley, CA, USA.

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|February 7, 2026
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Summary
This summary is machine-generated.

Researchers developed RNA-guided trans-splicing with Cas editor (RESPLICE) for transient RNA editing. This method efficiently and specifically inserts RNA cargo into endogenous transcripts, offering precise control over cellular functions with minimal off-target effects.

Keywords:
CRISPRCas13Cas7-11RNA editingalternative splicingchimeric RNAsexon replacementmolecular engineeringsynthetic biologytrans-splicing

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Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • RNA Therapeutics

Background:

  • RNA editing offers transient cellular alteration with low off-target risk compared to genome editing.
  • Existing exon-skipping technologies primarily influence splice site selection.
  • Many transcriptomic modifications require exogenous exon addition or replacement for therapeutic applications.

Purpose of the Study:

  • To develop a novel RNA editing technology for precise transcriptome modification.
  • To enable the replacement or addition of exogenous exons to target messenger RNAs (mRNAs).
  • To engineer transient and programmable control over cellular functions via RNA manipulation.

Main Methods:

  • Development of RNA-guided trans-splicing with Cas editor (RESPLICE).
  • Utilized two orthogonal RNA-targeting CRISPR effectors for co-localization and cis-splicing inhibition.
  • Demonstrated RNA cargo insertion into endogenous transcripts across multiple cell types.

Main Results:

  • Achieved efficient, specific, and programmable trans-splicing of RNA cargo (up to 2.1 kb).
  • Successfully targeted 11 endogenous transcripts in 3 different cell types.
  • Demonstrated up to 45% trans-splicing efficiency in bulk populations and 90% in sorted cells with high effector expression.

Conclusions:

  • RESPLICE represents a novel mode of RNA editing.
  • This technology enables fine-tuned and transient control of cellular programs.
  • RESPLICE offers a promising tool for RNA-based therapeutic strategies and biological research.