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2024

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NAR | Tsinghua University Zhang Qiyi team designed and verified a CRISPR activation system and anti-CRISPR protein to establish adaptability in mammalian cells

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A central challenge in the search for precise gene regulation in mammalian cells is the development of regulatory networks that enable perfect adaptation-in which the output continuously returns to a set post-stimulus baseline.

On August 1, 2024, the Tsinghua University Zhang Syu team was inNucleic Acids Research Published online entitled“CRISPR perfect adaptation for robust control of cellular immune and apoptotic responses”research paper, the studySuch a system is proposed to use CRISPR activation (CRISPRa) and anti-CRISPR proteins as two opposing elements to establish perfect adaptability in mammalian cells and dynamically regulate gene expression.

The research proves that the system can maintain the stable expression level of the target gene in the face of external disturbance, thus providing a powerful platform for biological applications. The versatility of the system is further demonstrated by its ability to integrate with endogenous regulatory mechanisms of T cells, such as NF-κb-mediated immune responses, and to program apoptotic responses with precise temporal and spatial control of cell growth and death.ResearchNot onlyThis has facilitated the understanding of gene regulation in mammalian cells and has opened up new avenues for therapeutic intervention, particularly in diseases characterized by dysregulated gene expression.

 

From feedback loops that control metabolic pathways to signal transduction networks that coordinate cells' responses to external stimuli, nature has evolved countless strategies to ensure stability or balance to counteract fluctuations and perturbations.This dynamic balance is essential not only for the proper functioning of biological pathways, but also for engineering new biological functions. Designing such a system would also, in turn, advance our understanding of the natural regulatory mechanisms that cells use to maintain homeostasis. In addition, adaptive synthetic biological systems hold important promise in the treatment of disease, possibly restoring homeostasis when genes are overexpressed or abnormally underexpressed, and provide new avenues for treating a myriad of diseases, including cancer, inflammation and neurodegenerative diseases.

By harnessing engineering principles, synthetic biology seeks to create artificial structures and genetic circuits that can replicate, improve, and ultimately control biological processes.The design of synthetic circuits that can achieve dynamic balance or perfect adaptation is an important feature of this field, that is, the condition that the system output returns to the baseline level after the transient response to the stimulus. There are two kinds of adaptive synthesis circuits widely used at present: incoherent feed-forward loops (iFFL) and negative feedback loops (NFLs). The iFFL consists of three parts: part A is activated in response to input, followed by downstream parts B and C, and part C inhibits part B from establishing iFFL. This cycle has been used in a variety of situations, such as controlling gene copy number variation and using CasE for resource control. However, iFFL requires precise suppression of dynamics to achieve robust control. Furthermore, modulation of endogenous genes using iFFL and CasE is challenging due to the need to insert specific hairpins in the target RNA sequence. In contrast, the key feature of NFL is its inhibitory feedback interaction, and gene expression control has been explored in mammalian cells.While simple NFL's are easy to achieve, they are difficult to restore to normal expression levels, which are essential for maintaining homeostasis.

Graphical Summary

In recent years, a more general feedback loop has been proposed, which can achieve both integral feedback and complete adaptation.At the heart of these systems are two opposing elements, Z1 and Z2. As long as the closed loop is stable, the concentration is stable without being affected by the endogenous pathway or any other perturbations, and finally a perfect adaptation is achieved. However, their use in mammalian cells is limited. Another strategy using sense/antisense messenger RNA (mRNA) suffers from challenges such as limited dynamic range, global translational inhibition, and the necessity to stabilize the mRNA. In addition, this realization may trigger a stress response within the cell due to the formation of double-stranded RNA. Given these challenges, the CRISPR system was proposed as an ideal framework for designing perfect adaptation mechanisms in mammalian cells. The emergence of anti-CRISPR (Acrs), which selectively inhibit CRISPR proteins, and their successful application in the mammalian environment, make CRISPR and Acrs ideal antithetical proteins. The versatility of single-guide RNA (sgRNA) design further enhances the system, allowing precise control of endogenous genes and facilitating disease therapeutic applications.
The study designed and validated a synthetic genetic circuit that uses the CRISPR activation (CRISPRa) system and anti-CRISPR proteins to establish perfect adaptation in mammalian cells.Closed-loop and open-loop configurations were constructed to control gene expression. In addition, the studied system can seamlessly integrate with endogenous regulatory mechanisms by designing sgRNAs against endogenous transcription factors (such as factors involved in NF-κb-mediated T cell immune responses), thereby providing intrinsic feedback without external intervention. Precise and stable regulation of the activity of immune cells in response to environmental stimuli has been achieved. In addition, the apoptotic response is programmed to ensure cellular adaptation, resulting in a controlled pattern of cell proliferation and death. The ability of the system to maintain stable gene expression levels under external interference demonstrates the robustness of biological applications and provides a new strategy for the treatment of diseases characterized by dysregulated gene expression.

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