se0c00034_si_001.pdf (1.29 MB)

CUT-LAMP: Contamination-Free Loop-Mediated Isothermal Amplification Based on the CRISPR/Cas9 Cleavage

Download (1.29 MB)
journal contribution
posted on 13.04.2020, 17:09 by Yijuan Bao, Yongzhong Jiang, Erhu Xiong, Tian Tian, Zhenzhen Zhang, Jing Lv, Yang Li, Xiaoming Zhou
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.