Universal and naked-eye gene detection platform based on CRISPR/Cas12a/13a system

Abstract

Colorimetric gene detection based on gold nanoparticles (AuNPs) is an attractive detection format due to its 1 simplicity. Here, we report a new design for a colorimetric gene-sensing platform based on the CRISPR/Cas system 2 that has improved specificity, sensitivity, and universality. CRISPR/Cas12a and CRISPR/Cas13a have two distinct 3 catalytic activities and are used for specific target gene recognition. Programmable recognition of DNA by 4 Cas12a/crRNA and RNA by Cas13a/crRNA with a complementary sequence activates the nonspecific trans-ssDNA or 5 -RNA cleavage, respectively, thus degrading the ssDNA or RNA linkers which are designed as a hybridization 6 template for the AuNP-DNA probe pair. Target-induced trans -ssDNA or RNA cleavage leads to a distance-dependent 7 color change for the AuNP-DNA probe pair. In this platform, naked eye detection of transgenic rice, African swine 8 fever virus (ASFV), and a miRNA can be completed within 1 hour. Our colorimetric gene-sensing method shows 9 superior characteristics, such as probe universality, isothermal reaction conditions, on-site detection capability, and 1 0 sensitivity that is comparable to that of the fluorescent detection; thus, this method represents a robust next generation 1 1 gene detection platform. 1 2

Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities

“…Copyright 2020 American Chemical Society C) Schematic representation of the Cas12a RNP and Cas13a RNP posing target-dependent trans-cleavage activity, resulting in a degradation of linker DNA, inhibiting AuNP aggregation. Reproduced with permission from ( Yuan et al, 2020 ). Copyright 2020 American Chemical Society.…”

“…Yuan et al adapted a AuNP-based colorimetric assay by incorporating Cas12a and Cas13a effector proteins for target amplification-free CRISPR sensing ( Yuan et al, 2020 ). Traditional AuNP colorimetric assays are based on hybridization between target nucleotides and nucleotides present on the AuNPs.…”

Cas12a-Based On-Site and Rapid Nucleic Acid Detection of African Swine Fever

“…Moreover, the RNA probe can generate false positive results because RNase is ever present and RNA is generally unstable (Gootenberg et al, 2017Myhrvold et al, 2018;Sashital, 2018;Shan et al, 2019). The newly reported naked-eye gene detection platform based on the CRISPR/Cas12a/13a system also needs a centrifugal machine and whole blood samples can interrupt the naked-eye detection (Yuan et al, 2019).…”

Cas12a-based on-site and rapid nucleic acid detection of African swine fever

“…Moreover, the RNA probe can generate false positive results because RNase is ever present and RNA is generally unstable 9, 11-13] . The newly reported naked-eye gene detection platform based on the CRISPR/Cas12a/13a system also needs a centrifugal machine and whole blood samples can interrupt the naked-eye detection [17] .…”