<br>利用 CRISPR-Cas12a 的特异性缺陷进行可编程多路核酸检测,Advanced Science

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利用 CRISPR-Cas12a 的特异性缺陷进行可编程多路核酸检测
Advanced Science ( IF 14.3 ) Pub Date : 2024-12-04 , DOI: 10.1002/advs.202411021
Xin Guan _{1,

2} , Rui Yang _{1,

2} , Jiongyu Zhang _{1,

2} , Jeong Moon ₁ , Chengyu Hou _{1,

2} , Chong Guo _{1,

2} , Lori Avery ₃ , Danielle Scarola ₄ , Daniel S Roberts ₄ , Rocco LaSala ₃ , Changchun Liu ₁

Affiliation

CRISPR-Cas12a 的工作原理类似于核酸检测中的复杂算法，但由于其特异性限制，它的挑战在于有时无法区分不匹配的目标。在这里，研究了 CRISPR-Cas12a 反应中错配的数量、位置和类型，并发现了其对错配的各种耐受性。利用 CRISPR-Cas12a 酶的特异性缺陷，设计了一种双模式检测策略，包括靶序列的近似匹配和精确查询，并开发了一种可编程的多重核酸检测。通过该检测，可同时检测到 14 种高危人瘤病毒（HPV）亚型，这些亚型共同占宫颈癌病例的 99%，具有阿摩尔敏感性。具体来说，该检测不仅区分了 HPV16 和 HPV18 这两种最常见的亚型，还检测了其他 12 种高危混合 HPV 亚型。为了实现低成本的即时检测，该检测被整合到基于纸张的微流体芯片中。此外，通过测试 75 个临床拭子样本验证了纸基微流控芯片的临床性能，达到了与 PCR 相当的性能。这种可编程的多重核酸检测试剂盒有可能广泛用于不同病原体的灵敏、特异性和同时检测。

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Programmable Multiplexed Nucleic Acid Detection by Harnessing Specificity Defect of CRISPR-Cas12a

CRISPR-Cas12a works like a sophisticated algorithm in nucleic acid detection, yet its challenge lies in sometimes failing to distinguish targets with mismatches due to its specificity limitations. Here, the mismatch profiles, including the quantity, location, and type of mismatches in the CRISPR-Cas12a reaction, are investigated and its various tolerances to mismatches are discovered. By harnessing the specificity defect of the CRISPR-Cas12a enzyme, a dual-mode detection strategy is designed, which includes approximate matching and precise querying of target sequences and develop a programmable multiplexed nucleic acid assay. With the assay, 14 high-risk human papillomavirus (HPV) subtypes are simultaneously detected, collectively responsible for 99% of cervical cancer cases, with attomolar sensitivity. Specifically, the assay not only distinguishes HPV16 and HPV18, the two most common subtypes but also detects 12 other high-risk pooled HPV subtypes. To enable low-cost point-of-care testing, the assay is incorporated into a paper-based microfluidic chip. Furthermore, the clinical performance of the paper-based microfluidic chip is validated by testing 75 clinical swab samples, achieving performance comparable to that of PCR. This programmable multiplexed nucleic acid assay has the potential to be widely applied for sensitive, specific, and simultaneous detection of different pathogens.

更新日期：2024-12-04

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