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Nitroreductase-Responsive Oligomeric crRNAs for Enzyme-Triggered Regulation of CRISPR Activity

Wen-Da Chen1,2, Li Liu1,2, Liang Cheng1,3,2

  • 1Beijing National Laboratory for Molecular Sciences (BNLMS), CAS Key Laboratory of Molecular Recognition and Function, CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China.

The Journal of Organic Chemistry
|March 12, 2026
PubMed

Insights

Scientists developed a new CRISPR system controlled by tumor-specific enzymes. This enzyme-responsive clustered regularly interspaced short palindromic repeats (CRISPR) system is activated only in hypoxic tumors, enabling targeted gene editing.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Gene Editing Technologies

Background:

  • Hypoxic tumors overexpress nitroreductase (NTR), offering a target for selective activation.
  • CRISPR-Cas systems offer precise gene editing but lack tumor-specific control.

Purpose of the Study:

  • To synthesize novel nitroreductase (NTR)-responsive clustered regularly interspaced short palindromic repeats (CRISPR) guide RNAs.
  • To establish a strategy for enzyme-regulated CRISPR control for targeted cancer therapy.

Main Methods:

  • Incorporation of a p-nitrobenzyl (p-NB) phosphoramidite at the 5' terminus of crRNAs.
  • Click-mediated oligomerization of crRNAs into trimeric and tetrameric constructs.
  • Enzymatic cleavage by NTR to release active crRNAs and restore Cas nuclease activity.

Main Results:

  • Constructed NTR-responsive CRISPR guide RNAs with suppressed Cas nuclease activity.
  • Demonstrated NTR-mediated cleavage of the linker, releasing active crRNAs.
  • Restored DNA cleavage activity of CRISPR-Cas systems *in vitro* upon NTR induction.

Conclusions:

  • Developed a method for synthesizing enzyme-responsive CRISPR guide RNAs.
  • Established a proof-of-concept for enzyme-regulated CRISPR control.
  • This strategy enables targeted CRISPR activation in hypoxic tumors via NTR.

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