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Photoactivatable CRISPR/Cas12a Strategy for One-Pot DETECTR Molecular Diagnosis
Analytical Chemistry ( IF 6.7 ) Pub Date : 2022-06-28 , DOI: 10.1021/acs.analchem.2c01193 Yong Chen 1, 2 , Xiaoling Xu 1 , Jiachun Wang 1 , Yibin Zhang 1 , Wentao Zeng 1 , Yizhen Liu 1 , Xueji Zhang 1
Analytical Chemistry ( IF 6.7 ) Pub Date : 2022-06-28 , DOI: 10.1021/acs.analchem.2c01193 Yong Chen 1, 2 , Xiaoling Xu 1 , Jiachun Wang 1 , Yibin Zhang 1 , Wentao Zeng 1 , Yizhen Liu 1 , Xueji Zhang 1
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As a golden partner of recombinase polymerase amplification (RPA), CRISPR/Cas12a has been proven to solve the false-positive problem caused by nonspecific amplification perfectly; meanwhile, its trans-cleave activity has further enhanced the sensitivity. However, the solution transfer operation after tube cap opening greatly increases the risk of aerosol contamination of amplicon, which is inconsistent with point-of-care (POC) diagnostics requirements. This study proposes a photoactivated CRISPR/Cas12a strategy to achieve one-pot high-sensitivity nucleic acid detection. Using photocleavable complementary ssDNA to block crRNA, RPA amplification can smoothly pass through the exponential interval without being affected by activated Cas12a in the critical early stage. After enough amplicons were produced, the Cas12a test was activated by short bursts of ultraviolet radiation at 365 nm. This one-pot method achieved a sensitivity of 2.5 copies within 40 min. This simple and sensitive one-pot method can effectively avoid amplicon contamination and lower the threshold for molecular diagnostics in POC.
中文翻译：
用于一锅检测分子诊断的光激活 CRISPR/Cas12a 策略
作为重组酶聚合酶扩增（RPA）的黄金搭档，CRISPR/Cas12a已被证明可以完美解决非特异性扩增导致的假阳性问题；同时，它的反式-cleave 活性进一步提高了灵敏度。然而，管盖打开后的溶液转移操作大大增加了扩增子气溶胶污染的风险，这与即时 (POC) 诊断要求不一致。本研究提出了一种光激活CRISPR/Cas12a策略，实现一锅高灵敏度核酸检测。使用光裂解互补ssDNA阻断crRNA，RPA扩增可以顺利通过指数区间，在关键早期不受活化Cas12a的影响。在产生足够的扩增子后，Cas12a 测试被 365 nm 的短脉冲紫外线辐射激活。这种一锅法在 40 分钟内达到了 2.5 个拷贝的灵敏度。
更新日期：2022-06-28
中文翻译：
用于一锅检测分子诊断的光激活 CRISPR/Cas12a 策略
作为重组酶聚合酶扩增（RPA）的黄金搭档，CRISPR/Cas12a已被证明可以完美解决非特异性扩增导致的假阳性问题；同时，它的反式-cleave 活性进一步提高了灵敏度。然而，管盖打开后的溶液转移操作大大增加了扩增子气溶胶污染的风险，这与即时 (POC) 诊断要求不一致。本研究提出了一种光激活CRISPR/Cas12a策略，实现一锅高灵敏度核酸检测。使用光裂解互补ssDNA阻断crRNA，RPA扩增可以顺利通过指数区间，在关键早期不受活化Cas12a的影响。在产生足够的扩增子后，Cas12a 测试被 365 nm 的短脉冲紫外线辐射激活。这种一锅法在 40 分钟内达到了 2.5 个拷贝的灵敏度。
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