Construction of tumor gene circuits using CRISPR/Cas tool and their applications

doi:10.12211/2096-8280.2021-046

Abstract

Abstract:

Refractory diseases such as malignant tumors are still a major challenge for public health in the world. In recent years, the fast development of synthetic biology has greatly promoted novel treatment strategies for diseases with great potentials for effective treatment. The artificially constructed gene circuits can specifically identify and distinguish diseased cells from normal cells, and control their fate to provide a new way for precise treatment. However, the ultimate challenge in constructing gene circuits is the lack of effective, programmable, safe, and sequence-specific gene editing tools. The clustered regularly interspaced short palindromic repeat (CRISPR) system and CRISPR-associated RNA-guided endonuclease Cas9-targeted (CRISPR-associated protein 9) genome editing tool have recently been applied in engineering gene circuits for its unique properties: manipulability, high efficiency and programmability. In addition to indel mutations induced by Cas9, CRISPR/Cas can perform DNA/RNA base editing, providing efficient tools for gene circuit design, and greatly improve efficiency for designing circuit elements. The traditional single-targeted treatment cannot effectively distinguish tumor cells from normal cells, and gene therapy has poor anti-tumor effects, which severely limits its application. Currently, the design of gene circuits using tumor-specific targets based on CRISPR/Cas systems provides a new strategy for precision cancer therapy. Scientists have developed a series of efficient and targeted transcription factor components based on CRISPR technology to maximize the performance of gene circuits. These novel designs extend the toolbox for gene editing, and enable the construction of intelligent gene circuits such as logic gates, signal conductors, analogue computing circuits, counters and memory devices. Therefore, the application of intelligent gene circuits based on CRISPR technology can effectively ensure safety, efficiency and specificity for cancer treatment. This article introduces the updated progress, prospects and potential challenges of CRISPR/Cas technology for the design and construction of gene circuits in biomedical field. Firstly, the efficacy of synthetic gene circuits using CRISPR system components in tumor treatment is evaluated, especially the safety and effectiveness of using Cas9 induced by artificial switch to intervene tumor growth. Secondly, the design of CRISPR/Cas9-mediated signal conductors is introduced, which can recognize multiple protein signals simultaneously and realize the simultaneous regulation of multiple genes. In addition, the CRISPR/Cas12a system with smaller size, strong specificity and high gene regulation efficiency is a new generation alternative. Finally, the simplified gene circuit design based on the promoter-free CRISPReader expression elements is addressed, and its high-efficiency startup feature will greatly enhance the potential application of intelligent gene circuits in precise cancer treatments in the future.

Key words: gene circuits, signal conductor, malignant tumors, clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-associated protein

摘要：

以恶性肿瘤为代表的难治性疾病仍是困扰我国乃至全球的一个重大公共卫生问题。近年来，合成生物学的蓬勃发展，极大推动了疾病创新治疗策略，并显示出巨大潜力。人工构建的基因线路可特异性地识别、区分疾病细胞和正常细胞，并控制疾病细胞命运，为精准治疗提供了新途径。然而构建基因线路用于疾病治疗的挑战之一是缺乏有效、可编程、安全和序列特异性的基因编辑工具。CRISPR/Cas自从首次被用于哺乳动物基因编辑以来，已被广泛应用于研究、工业和医学领域。除Cas9诱导的indel突变外，CRISPR/Cas能够进行DNA/RNA的碱基编辑，为基因线路设计提供了高效的工具，极大提高了每个线路元件效率。因此，基于CRISPR技术的智能化基因线路的应用，可有效保证癌症等疾病治疗的安全性、高效性和特异性。本文介绍了CRISPR/Cas技术应用于医学合成生物学基因线路设计的研究进展和潜在挑战。评估了使用CRISPR系统元件的合成基因线路在肿瘤治疗中的效果，尤其利用人工开关诱导的Cas9干预肿瘤细胞治疗的安全性和有效性；介绍了CRISPR/Cas9介导的信号传感器设计，其可同时识别多个蛋白质信号，实现了多基因同步调控，以及新一代体系小、特异性强、基因调控效率高的CRISPR/Cas12a系统及其遗传改造。基于无启动子表达CRISPReader元件的精简基因线路设计，其高效启动的特点极大提升了智能化基因线路在未来精准癌症治疗中的应用潜力。

关键词: 基因线路, 信号传感器, 恶性肿瘤, 成簇的规则间隔短回文重复, CRISPR相关蛋白

CLC Number:

Q812

Fei SONG, Yuchen LIU, Zhiming CAI, Weiren HUANG. Construction of tumor gene circuits using CRISPR/Cas tool and their applications[J]. Synthetic Biology Journal, 2022, 3(1): 53-65.

宋斐, 刘宇辰, 蔡志明, 黄卫人. 基于CRISPR/Cas工具的肿瘤基因线路构建及应用[J]. 合成生物学, 2022, 3(1): 53-65.

Figures/Tables 4

Fig. 1 Schematic representation for the artificial gene circuits developed based on CRISPR/Cas technology(a) artificial sequences of transcription factor binding site are inserted into the upstream of the Cas9 coding sequences to control gene expression by sensing intracellular signal proteins. In malignant tumor cells, some specific transcription factors such as β-catenin and NF-κB are abnormally activated. The expression of the downstream CRISPR/Cas genes is turned on when abnormal signal proteins in malignant cells bind to the transcription factor binding site and RNA polymerase (RNA poly) is recruited to the TATA box. Effector represents the Cas9 protein. (b) the light-inducible gene expression regulation system in cancer cells developed based on CRISPR/Cas9 technology. Blue light stimulation induces heterodimerization between A. thaliana cryptochrome 2 (CRY2) and its binding partner CIBN (cryptochrome-interacting basic helix-loop-helix protein 1). Therefore, the transcriptional activation domain (AD) fused with the CRY2 protein is targeted to the specific region and promotes the expression of downstream genes. (c) schematic diagram for the light inducible CRISPR/dCas9 system. The dCas9 is split into two fragments lacking nuclease activity, and the dCas9 fragments are fused with light-inducible dimerization domains (pMag and nMag). Blue light stimulation induces heterodimerization between pMag and nMag, which enables split dCas9 fragments to reassociate, thereby reconstituting RNA-guided transcriptional activation activity. (d) schematic diagram for a small molecular artificial switch system. The binding of doxycycline results in the conformational change of reverse tetracycline transcriptional activator rtTA, and then the activated rtTA could bind to the Tet-responsive element (TRE) to drive the expression of the target genes, such as Cas9 gene.

Fig. 2 Schematic diagram for the signal conductor that links one signal with another developed based on CRISPR/Cas technology[The β-catenin activates the Wnt pathway, and promotes the proliferation of tumor cells. The redesigned sgRNA preferentially binds to the endogenous β-catenin, and then couples with dCas9-AD protein to activate the output genes, such as the endogenous tumor suppressor genes (eg, p53) or apoptosis genes (eg, p21 and caspase 3), enabling the tumor cells to redirect oncogenic signaling to an anti-oncogenic pathway.]

Fig. 3 CRISPReader drives gene expression by coupling the transcriptional and translational mechanisms［52］(a) CRISPReader is constructed by combining transcriptional and translational platforms. The dCas9-VP64 protein robustly activated transcription of reporter is constructed when combined with sgRNA targeting sequences near the TATA box. Then, the RNA activator leads to the formation of initiation factor complexes involving eIF4G and recruits ribosomes to initiate translation. (b) mechanisms of the CRISPReader designed to drive the gene cluster expression. After dCas9-VP64-mediated transcription, the RNA activators bind to each targeted mRNA and independently initiates mRNA translation.

Fig. 4 Design and construction of the AND gate minigene circuits［54］(The UPII promoter drives the transcription of Cas9 mRNA, while the TERT promoter is used to promote the transcription of sgRNA targeting LacI. The output Renilla luciferase gene is regulated by a LacI-controlled CMV promoter. The luciferase is expressed only when both UPII promoter and TERT promoter are activated. In the design of the minigene circuit, the UPII and TERT promoters are replaced by their respective transcription factor binding elements. Both c-Myc and Get1, only in bladder cancer cells, have a relative high expression level at the same time. After initial expression of sgRNA1 and sgRNA2, they could further bind to the upstream of their own transcription initiation sites, and amplify the transcription signals of c-Myc and Get1 through the positive feedback mechanism to amplify their downstream gene transcription. Furthermore, the LacI gene is knocked out by sgRNA2, and luciferase reporter gene is activated by transcription. In normal bladder epithelial cells, luciferase could not be effectively transcribed and further silenced by a trace amount of LacI expressed at the background level.)

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