College of Biophotonics & School of Life Sciences, South China Normal University, Guangzhou 510631, P. R. China.
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P. R. China.
ACS Sens. 2020 Apr 24;5(4):1082-1091. doi: 10.1021/acssensors.0c00034. Epub 2020 Apr 9.
Loop-mediated isothermal amplification (LAMP) is a sensitive and widely used gene amplification technique. However, high amplification efficiency and amplification products containing multiple inverted repeats make the LAMP reaction extremely vulnerable to false-positive amplification caused by contamination. Herein, a contamination-free LAMP (CUT-LAMP) assisted by the CRISPR/Cas9 cleavage with superior reliability and durability has been reported. The core of CUT-LAMP is the engineering of the forward or backward inner primer in the target-independent region, which makes the LAMP products contain a protospacer adjacent motif (PAM) site for the CRISPR/Cas9 recognition. For the CUT-LAMP reaction, cross-contamination can be efficiently cleaved by the corresponding Cas9/sgRNA, but the target gene can get rid of digestion due to the lack of a PAM site near the recognition region. CUT-LAMP shows impressive contamination resistance but does not significantly increase procedure complexity; thus, it represents a simple and versatile toolkit facilitating the adoption by open- and closed-tube detection format.
环介导等温扩增 (LAMP) 是一种灵敏且广泛应用的基因扩增技术。然而，高扩增效率和扩增产物中含有多个反向重复序列，使得 LAMP 反应极易受到污染引起的假阳性扩增的影响。在此，我们报道了一种基于 CRISPR/Cas9 切割的无污染 LAMP（CUT-LAMP），具有卓越的可靠性和耐用性。CUT-LAMP 的核心是在目标非依赖性区域对正向或反向内引物进行工程改造，这使得 LAMP 产物包含一个间隔相邻基序 (PAM) 位点，可被 CRISPR/Cas9 识别。对于 CUT-LAMP 反应，交叉污染可以被相应的 Cas9/sgRNA 有效切割，但由于识别区域附近缺乏 PAM 位点，目标基因可以逃避消化。CUT-LAMP 表现出令人印象深刻的抗污染能力，但不会显著增加程序复杂性；因此，它代表了一种简单且通用的工具包，便于采用开管和闭管检测格式。
