Sensitive osteosarcoma diagnosis through five-base telomerase product-triggered CRISPR-Cas12a enhanced rolling circle amplification,Analytical Methods

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Sensitive osteosarcoma diagnosis through five-base telomerase product-triggered CRISPR-Cas12a enhanced rolling circle amplification
Analytical Methods ( IF 2.7 ) Pub Date : 2021-08-02 , DOI: 10.1039/d1ay00952d
Xing Zhou ₁ , Jun-Liang Zhang ₁ , Meng-Han Chang ₁ , Gen-Tao Fan ₁ , Xiao-Zhou Liu ₁ , Su-Jia Wu ₁ , Xin Shi ₁

Affiliation

Osteosarcoma is the most frequent primary malignant bone tumor, composed of mesenchymal cells producing osteoid and immature bone. The sensitive detection of telomerase plays a pivotal role in the early diagnosis and therapeutic treatment of osteosarcoma. We report here an in vitro strategy for sensitive telomerase activity detection through the integration of rolling circle amplification (RCA) and a clustered regularly spaced short palindrome repeats (CRISPR)-Cas12a system. In the proposed strategy, telomerase substrate (TS) primers are easily controlled to extend five bases (GGGTT) to give short telomerase extension products (TEP) with definite lengths without adding dATP. The resulting short TEPs can then cyclize the padlock through hybridizing with its two terminals and thus initiate the following RCA. To obtain an improved sensitivity, the CRISPR-Cas12a system is attached to collaterally cut surrounding DNA reporter probes after recognizing the target single strand DNA sequence in the RCA products. The highlights of this strategy are as follows: (i) the short TEP triggered strategy is excellent at detecting low telomerase activity and thus contributes to the early diagnosis of malignant tumors; (ii) highly sensitive telomerase activity detection which is easy to operate from RCA initiated CRISPR-Cas12a; (iii) opening up of a new avenue for telomerase activity detection with a CRISPR-Cas12a system. Finally, the proposed strategy exhibited sensitive telomerase activity detection under optimized experimental parameters and has great application potential for the clinical diagnosis of malignant tumors and the development of anti-cancer drugs.

中文翻译：

通过五碱基端粒酶产物触发的 CRISPR-Cas12a 增强滚环扩增进行敏感骨肉瘤诊断

骨肉瘤是最常见的原发性恶性骨肿瘤，由产生类骨质和未成熟骨的间充质细胞组成。端粒酶的灵敏检测在骨肉瘤的早期诊断和治疗中起着举足轻重的作用。我们在这里报告了一个体外通过整合滚环扩增 (RCA) 和成簇的规则间隔短回文重复序列 (CRISPR)-Cas12a 系统进行灵敏端粒酶活性检测的策略。在所提出的策略中，端粒酶底物 (TS) 引物很容易控制以延长五个碱基 (GGGTT)，从而在不添加 dATP 的情况下提供具有确定长度的短端粒酶延伸产物 (TEP)。由此产生的短 TEP 然后可以通过与挂锁的两个终端杂交来环化挂锁，从而启动以下 RCA。为了获得更高的灵敏度，CRISPR-Cas12a 系统在识别出 RCA 产品中的目标单链 DNA 序列后，连接到旁切的周围 DNA 报告探针。该策略的亮点如下：(i)短TEP触发策略在检测低端粒酶活性方面表现出色，从而有助于恶性肿瘤的早期诊断；(ii) 从RCA启动的CRISPR-Cas12a易于操作的高灵敏度端粒酶活性检测；(iii) 使用 CRISPR-Cas12a 系统为端粒酶活性检测开辟了一条新途径。最后，所提出的策略在优化的实验参数下表现出灵敏的端粒酶活性检测，在恶性肿瘤的临床诊断和抗癌药物的开发中具有巨大的应用潜力。(iii) 使用 CRISPR-Cas12a 系统为端粒酶活性检测开辟了一条新途径。最后，所提出的策略在优化的实验参数下表现出灵敏的端粒酶活性检测，在恶性肿瘤的临床诊断和抗癌药物的开发中具有巨大的应用潜力。(iii) 使用 CRISPR-Cas12a 系统为端粒酶活性检测开辟了一条新途径。最后，所提出的策略在优化的实验参数下表现出灵敏的端粒酶活性检测，在恶性肿瘤的临床诊断和抗癌药物的开发中具有巨大的应用潜力。

更新日期：2021-08-27

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