A spacer design strategy for CRISPR‐Cas12f1 with single‐nucleotide polymorphism mutation resolution capability and its application in the mutations diagnosis of pathogens

Abstract Infectious diseases remain a major global issue in public health. It is important to develop rapid, sensitive, and accurate diagnostic methods to detect pathogens and their mutations. Cas12f1 is an exceptionally compact RNA‐guided nuclease and have the potential to fulfill the clinical needs. Based on the interaction between crRNA‐SSDNA binary sequence and Cas12f1, here, we addressed the essential features that determine the recognition ability of CRISPR‐Cas12f1 single‐nucleotide polymorphism (SNP), such as the length of spacer region and the base pairing region that determines the trans‐cleavage of ssDNA. A fine‐tuning spacer design strategy is also proposed to enhance the SNP recognition capability of CRISPR‐Cas12f1. The optimized spacer confers the Cas12f1 system a strong SNP identification capability for viral or bacterial drug‐resistance mutations, with a specificity ratio ranging from 19.63 to 110.20 and an admirable sensitivity up to 10 0 copy/μL. Together, the spacer screening and CRISPR‐Cas12f1 based SNP identification method, is sensitive and versatile, and will have a wide application prospect in pathogen DNA mutation diagnosis and other mutation profiling.