A PAM-free CRISPR/Cas12a ultra-specific activation mode based on toehold-mediated strand displacement and branch migration

You Wu ¹, Wang Luo ¹, Zhi Weng ¹, Yongcan Guo ², Hongyan Yu ¹, Rong Zhao ¹, Li Zhang ¹, Jie Zhao ³, Dan Bai ¹, Xi Zhou ¹, Lin Song ¹, Kena Chen ¹, Junjie Li ¹, Yujun Yang ¹, Guoming Xie ¹

Abstract

CRISPR (clustered regularly interspaced short palindromic repeats) technology has achieved great breakthroughs in terms of convenience and sensitivity; it is becoming the most promising molecular tool. However, only two CRISPR activation modes (single and double stranded) are available, and they have specificity and universality bottlenecks that limit the application of CRISPR technology in high-precision molecular recognition. Herein, we proposed a novel CRISPR/Cas12a unrestricted activation mode to greatly improve its performance. The new mode totally eliminates the need for a protospacer adjacent motif and accurately activates Cas12a through toehold-mediated strand displacement and branch migration, which is highly universal and ultra-specific. With this mode, we discriminated all mismatch types and detected the EGFR T790M and L858R mutations in very low abundance. Taken together, our activation mode is deeply incorporated with DNA nanotechnology and extensively broadens the application boundaries of CRISPR technology in biomedical and molecular reaction networks.