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Reining in Cas13a activity with N-terminal removable tags expands Cas13a based molecular sensing and enables precise

Wenjun Liu1, Xuena Zhu2, Ling Li3

  • 1Zhejiang Provincial Key Laboratory of Pancreatic Disease, The First Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, 310003, China; Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, 310003, China; The Innovation Center for the Study of Pancreatic Diseases of Zhejiang Province, Zhejiang University Cancer Center, Hangzhou, 310003, China.

Biosensors & Bioelectronics
|February 22, 2023
PubMed

Insights

Researchers developed a novel controlled activation system for Cas13a using N-terminus tagging. This system enables precise gene knockdown and protease detection by regulating Cas13a activity with SUMO tags and proteases.

Area of Science:

  • Molecular Biology
  • Biotechnology
  • Gene Editing Tools

Background:

  • Cas13 enzymes are RNA-guided RNases activated by target binding, leading to collateral cleavage of non-target RNAs.
  • This collateral activity is utilized in gene interference and biosensing applications.
  • Controlling Cas13 activation remains a challenge for precise temporal and spatial applications.

Purpose of the Study:

  • To design and validate a novel multi-component system for controlled activation of Cas13a.
  • To develop a Cas13a-based biosensor for protease detection.
  • To enable targeted gene knockdown in specific cell types based on protease levels.

Main Methods:

  • Engineered a composite SUMO tag (His, Twinstrep, Smt3) fused to the N-terminus of Cas13a to suppress its activity.
  • Investigated protease-mediated cleavage of the SUMO tag to release Cas13a activity.
  • Developed a biosensor utilizing SUMO-Cas13a to detect the protease Ulp1.
  • Programmed SUMO-Cas13a for targeted gene knockdown in cells with high SUMO protease expression.

Main Results:

  • The composite SUMO tag effectively suppressed target-dependent activation of Cas13a by interfering with crRNA binding.
  • Protease-mediated cleavage of the tag restored Cas13a activity, allowing for controlled activation.
  • The SUMO-Cas13a biosensor demonstrated a limit of detection (LOD) of 48.8 pg/μL for Ulp1.
  • Achieved preferential target gene knockdown in cells expressing high levels of SUMO protease.

Conclusions:

  • A novel regulatory system for Cas13a activation based on N-terminus SUMO tagging was successfully developed.
  • This system enables Cas13a-based protease detection for the first time.
  • The strategy provides a new method for achieving temporal and spatial control over Cas13a activity for therapeutic and diagnostic applications.
Keywords:
CRISPRCas13Collateral cleavageMulti-component regulationProteaseSUMO

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