Colorimetric gene detection based on gold nanoparticles (AuNPs) is an attractive detection format due to its simplicity. Here, we report a new design for a colorimetric gene-sensing platform based on the CRISPR/Cas system that has improved specificity, sensitivity, and universality. CRISPR/Cas12a and CRISPR/Cas13a have two distinct catalytic activities and are used for specific target gene recognition. Programmable recognition of DNA by Cas12a/crRNA and RNA by Cas13a/crRNA with a complementary sequence activates the nonspecific trans-ssDNA or -RNA cleavage, respectively, thus degrading the ssDNA or RNA linkers which are designed as a hybridization template for the AuNP-DNA probe pair. Target-induced trans -ssDNA or RNA cleavage leads to a distance-dependent color change for the AuNP-DNA probe pair. In this platform, naked eye detection of transgenic rice, African swine fever virus (ASFV), and a miRNA can be completed within 1 hour. Our colorimetric gene-sensing method shows superior characteristics, such as probe universality, isothermal reaction conditions, on-site detection capability, and sensitivity that is comparable to that of the fluorescent detection; thus, this method represents a robust next generation gene detection platform.
The recently reported freezing-based labeling method for constructing DNA-AuNP probes is rapid but still requires thiol modification. Here, we evaluated a poly(A)-tagged DNA sequence using the freezing-based labeling method, and the results demonstrated that approximately 10 A bases at the sequence ends are essential. More detailed observations revealed that some DNA sequences tend to form secondary structures and thus shield exposed A bases, resulting in inefficient or failed labeling. However, successful labeling was restored by simply increasing the poly(A)-base number. Building on these discoveries, we developed three kinds of AuNP-based bioprobes, DNA-AuNP, RNA-AuNP, and DNA-enzyme-AuNP, using the freezing-based labeling method. This method was completed in a single mixing step with no need for thiol modification, representing one of the most convenient and lowest cost AuNP bioprobe labeling techniques ever reported. In addition, the resulting AuNP bioprobes were further used to advance CRISPR-based diagnostics through the development of user-friendly colorimetric, fluorescence, and lateral flow detection strategies.
Stripe (yellow) rust, caused by Puccinia striiformis f. sp. tritici (Pst), can significantly affect wheat production. Cloning resistance genes is critical for efficient and effective breeding of stripe rust resistant wheat cultivars. One resistance gene (Yr10CG) underlying the Pst resistance locus Yr10 has been cloned. However, following haplotype and linkage analyses indicate the presence of additional Pst resistance gene(s) underlying/near Yr10 locus. Here, we report the cloning of the Pst resistance gene YrNAM in this region using the method of sequencing trait-associated mutations (STAM). YrNAM encodes a non-canonical resistance protein with a NAM domain and a ZnF-BED domain. We show that both domains are required for resistance. Transgenic wheat harboring YrNAM gene driven by its endogenous promoter confers resistance to stripe rust races CYR32 and CYR33. YrNAM is an ancient gene and present in wild wheat species Aegilops longissima and Ae. sharonensis; however, it is absent in most wheat cultivars, which indicates its breeding value.
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
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