image: Specific crRNAs can activate Cas13a even in the absence of a target RNA, resulting in robust, target-free RNA cleavage—hallmarks of RINCA
Credit: ©Science China Press
The complexity behind Cas13a activation
Cas13a consists of two lobes—the recognition (REC) and nuclease (NUC) domains—and is guided by a crRNA (50–58 nt) comprising a direct repeat sequence followed by a spacer region. Under the canonical model, the spacer hybridizes with a complementary RNA target to form a duplex that threads through a positively charged channel within the NUC lobe. This triggers conformational changes that activate the HEPN domains, enabling target cleavage (cis-cleavage) (Abudayyeh et al., Science, 2016).
Remarkably, activated Cas13a can also cleaves bystander RNAs through "trans-cleavage," a sequence-independent process extensively leveraged forli in vitro diagnostic (East-Seletsky et al., Nature, 2016). Intriguingly, recent reports also documented basal RNase activity in Cas13a even in the absence of both crRNA and RNA target, suggesting intrinsic enzymatic potential (Li et al., Nat. BME, 2024).
Cas13a activated by crRNA alone: a paradigm shift
Contrary to canonical models, the researchers found that specific crRNAs can activate Cas13a even in the absence of a RNA target, resulting in robust, bystander RNA cleavage—hallmarks of RINCA. This activity was confirmed using both gel-based and FRET assays and was shown to be crRNA sequence-dependent. Screening a crRNA library revealed that RINCA is surprisingly common among Cas13a guides.
Mechanistically, RINCA correlates with the predicted secondary structure of the crRNA spacer. crRNAs with structured spacers—particularly those forming stable stem regions—were more likely to trigger activation. Disrupting these structures reduced RINCA activity, while large loop formations abolished it entirely. By designing ligand-responsive aptamer-crRNAs, the team demonstrated that RINCA could be predictably turned on or off through spacer structure modulation.
RINCA functions inside cells and alters endogenous RNA stability
Kinetic modeling estimated that RINCA operates at about 30% the catalytic efficiency of canonical, target-dependent activation. Despite this reduced potency, binary Cas13a/str-crRNA RNP complexes were still active in mammalian cells. This unexpected activity led to decreased cell proliferation, widespread RNA degradation, and transcriptome shifts—particularly in RNA processing pathways—suggesting a cellular stress response to endogenous RNA loss.
Engineering safer Cas13a variants
RINCA presents a challenge for Cas13a-based biosensors and therapeutics by introducing unwanted background activity and potential cytotoxicity. To address these issues, the team rationally designed Cas13a mutants by targeting residues involved in crRNA interaction. Although most mutations either impaired all activity or had little effect, a distinct subset selectively suppressed RINCA without disrupting canonical RNA targeting. These optimized variants significantly reduced background signals in vitro and improved detection sensitivity. In cells, they also markedly reduced off-target RNA cleavage while preserving effective on-target gene knockdown.
Turning a bug into a feature: RINCA for cancer therapy
Beyond its characterization as an unintended activity, the researchers repurposed RINCA as a programmable therapeutic switch. By engineering ATP-responsive crRNAs, they created Cas13a RNP complexes that selectively activate in high-ATP cells—a hallmark of malignant cells. In both 2D and 3D HEK293 cell culture models, these systems markedly suppressed cell growth. In xenograft mice, they prevented tumor formation. In a hydrodynamic tail vein injection model of hepatocellular carcinoma (HCC), the system reduced tumor burden, restored liver structure, and normalized liver-to-body weight ratios.. Importantly, systemic safety assessments found no signs of toxicity, organ damage, or aberrant heptic or hematological markers, underscoring the therapeutic potential of RINCA-based platforms for precise cancer treatment with minimal side effects.
About the research team
This study was led by Dr. Wenjun Liu and Dr. Xuena Zhu (Assistant Professors), and Dr. Tingbo Liang (Professor and President) at The First Affiliated Hospital of Zhejiang University School of Medicine (FAHZU). The group specializes in cancer diagnostics and therapeutics, with a research emphasis on the mechanistic exploration and engineering of CRISPR-Cas systems for enhanced safety and precision. The work was carried out in close collaboration with Dr. Chen-Zhong Li (Professor and F-CAE) at The Chinese University of Hong Kong, Shenzhen.
Journal
Science Bulletin