使用 CRISPR-Cas12a 变体增强反式裂解活性,以减少顺式裂解产物的立体抑制。

IF 10.7 1区 生物学 Q1 BIOPHYSICS
Biosensors and Bioelectronics Pub Date : 2025-01-01 Epub Date: 2024-10-16 DOI:10.1016/j.bios.2024.116859
Injoo Hwang, Yo Han Song, Sanghwa Lee
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引用次数: 0

摘要

CRISPR-Cas12a系统因其无差别的反式ssDNase活性而成为一种前景广阔的分子诊断工具。然而,如果没有环介导等温扩增等前置扩增步骤,基于Cas12a的诊断灵敏度仍不足以用于临床,因此需要增强Cas12a的反式裂解活性。在这里,我们提出了一种新策略,通过减少顺式裂解产物的立体阻碍来增强 Cas12a 的反式裂解活性。我们设计了靶链加载(TSL)结构域中存在丙氨酸突变的 Cas12a 变体,从而降低了催化位点对靶链(TS)悬垂的亲和力,并将反式裂解效率显著提高了 5.8 倍。此外,我们还使用了一种新颖的盐稀释方法,利用 Cas12a 在低离子强度条件下增强的反式裂解活性(7 倍),显著提高了基于 Cas12a 的检测系统的灵敏度。为了证明基于 Cas12a 的检测系统的临床潜力,我们结合使用 KE1096AA Cas12a 突变体和盐稀释法,验证了该系统检测少量乙型肝炎病毒 (HBV) DNA 模型的能力。我们增强Cas12a反式裂解活性的策略为开发更灵敏、更高效的基于Cas12a的诊断方法铺平了道路。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Enhanced trans-cleavage activity using CRISPR-Cas12a variant designed to reduce steric inhibition by cis-cleavage products.

The CRISPR-Cas12a system has emerged as a promising tool for molecular diagnostics due to its indiscriminate trans-ssDNase activity. However, the sensitivity of Cas12a-based diagnostics remains insufficient for clinical use without a pre-amplification step such as loop-mediated isothermal amplification, and therefore the trans-cleavage activity of Cas12a needs to be enhanced. Here, we present a novel strategy to enhance the trans-cleavage activity of Cas12a by reducing the steric hindrance from cis-cleavage products. We have designed Cas12a variants with alanine mutations in the target strand loading (TSL) domain, resulting in reduced affinity for target strand (TS) overhangs to the catalytic site and significantly increased trans-cleavage efficiency by up to 5.8-fold. In addition, we used a novel salt dilution method to exploit the enhanced trans-cleavage activity of Cas12a under low ionic strength conditions (7-fold), significantly improving the sensitivity of our Cas12a-based detection system. To demonstrate the clinical potential of our Cas12a-based detection system, we validated its ability to detect small amounts of hepatitis B virus (HBV) DNA model using the combination of the KE1096AA Cas12a mutant and the salt dilution method, which enables the detection of DNA at atto-molar concentrations. Our strategy to enhance the trans-cleavage activity of Cas12a paves the way for the development of more sensitive and efficient Cas12a-based diagnostics.

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来源期刊
Biosensors and Bioelectronics
Biosensors and Bioelectronics 工程技术-电化学
CiteScore
20.80
自引率
7.10%
发文量
1006
审稿时长
29 days
期刊介绍: Biosensors & Bioelectronics, along with its open access companion journal Biosensors & Bioelectronics: X, is the leading international publication in the field of biosensors and bioelectronics. It covers research, design, development, and application of biosensors, which are analytical devices incorporating biological materials with physicochemical transducers. These devices, including sensors, DNA chips, electronic noses, and lab-on-a-chip, produce digital signals proportional to specific analytes. Examples include immunosensors and enzyme-based biosensors, applied in various fields such as medicine, environmental monitoring, and food industry. The journal also focuses on molecular and supramolecular structures for enhancing device performance.
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