Multilayer regulation of CRISPR systems: integrating anti-CRISPR proteins, CRISPRi/a, and quorum sensing networks.

The CRISPR-Cas system has evolved into a highly efficient platform for genome editing and programmable gene regulation, demonstrating broad application potential in microbiology, biotechnology, and medicine. However, traditional CRISPR tools typically rely on constitutively active nuclease activity; this constant activation state is prone to off-target effects and cytotoxicity, and lacks precise spatiotemporal regulation in complex biological environments. Therefore, developing strategies to achieve fine-tuned and context-dependent regulation of CRISPR activity has become a critical issue in this field that urgently needs to be addressed. Recent studies have demonstrated that various endogenous and exogenous regulatory modules can modulate the activity of the CRISPR-Cas system at different biological levels. Among these, anti-CRISPR proteins (Acr), which are natural inhibitory factors derived from bacteriophages, can suppress the nuclease activity of Cas proteins at the protein level by directly interfering with their function; The CRISPR interference/activation (CRISPRi/a) system, on the other hand, relies on catalytically inactivated Cas proteins to achieve sequence-specific regulation of target gene transcription; furthermore, quorum sensing (QS) networks dynamically regulate the expression of relevant genes by sensing cell density and environmental signals, thereby influencing the functional state of the CRISPR system at the population level. Based on the aforementioned regulatory mechanisms, this paper provides a comprehensive, literature-based overview of the molecular basis and recent advances in the applications of Acr proteins, the CRISPRi/a system, and QS networks in CRISPR-Cas regulation. Building on this, we propose a hierarchical regulatory framework: QS networks serve as upstream environmental sensing modules that drive CRISPRi/a-mediated programmable transcriptional regulation, while Acr proteins act as downstream rapid-response elements that finely tune CRISPR activity. This multi-tiered regulatory system holds promise for the dynamic optimization and precise control of CRISPR systems, offering new design concepts for constructing adaptive, programmable genetic regulatory networks, and demonstrating significant application potential in fields such as microbial engineering, anti-infective strategies, and precision gene regulation. The regulatory mechanism of Acr proteins on CRISPR activity has been experimentally validated in several studies. Nevertheless, the integration of Acr proteins with other regulatory modules such as CRISPRi/a systems or QS networks remains in the exploratory stage and requires further empirical research to confirm their functionality in complex biological systems.