Related Experiment Video
Updated: Jun 19, 2026
09:51
Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
Published on: May 25, 2018
Guide RNA reprogramming facilitates minimized tracrRNA-dependent off-target and versatile CRISPR/Cas9 engineering.
Wenxia Yu1,2, Jun Chen3, Junfan Guo4
1Department of Plastic and Reconstructive Surgery, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; Shanghai Institute of Precision Medicine, Shanghai, China.
Nature Communications
|June 17, 2026
Summary
Researchers found that guide RNAs (gRNAs) can be reprogrammed, leading to tracrRNA-dependent off-target effects. They developed new CRISPR-Cas9 systems to minimize these effects and enhance safety for gene editing applications.
Area of Science:
- Molecular Biology
- Gene Editing Technologies
- Biotechnology
Background:
- Current CRISPR-Cas9 systems exhibit safety concerns, including sequence-independent off-targeting.
- The guide RNA (gRNA) structure, specifically the crRNA:tracrRNA duplex, is splittable and reprogrammable.
- Endogenous RNAs can hijack gRNAs, causing tracrRNA-dependent off-target (TDO) effects.
Purpose of the Study:
- To address safety concerns and practical limitations in CRISPR-Cas9 systems.
- To investigate and mitigate tracrRNA-dependent off-target effects.
- To develop enhanced CRISPR platforms for improved safety and expanded applications.
Main Methods:
- Machine learning models trained on gRNA variant screens to establish optimal gRNA design rules.
- Engineering of crRNA variants mismatched to human/mouse transcriptomes to minimize TDO.
- Development of reprogrammable tracrRNAs for CRISPR activation (CRISPRa) and redesigned scaffolds to prevent PAM-less Cas9 self-editing.
Main Results:
- Identification of a novel off-target mechanism mediated by endogenous RNA hijacking of gRNAs.
- Successful engineering of crRNA variants that significantly reduce TDO effects.
- Creation of a separately expressed gRNA (segRNA) platform enabling multiplexed gene editing and functional annotation.
Conclusions:
- The splittability and reprogrammability of gRNAs present a previously unrecognized off-target risk.
- Developed strategies effectively minimize TDO, enhancing CRISPR system safety.
- The novel segRNA platform and reprogrammable tracrRNAs offer versatile tools for advanced gene editing and cellular research.
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