Universal and Naked-Eye Gene Detection Platform Based on the Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a/13a System

Abstract

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.

CRISPR‐Cas system for biomedical diagnostic platforms

“…(G‐H) Representative results for orthogonal identification of each bacterium. Reprinted with permission from American Chemical Society 66 . SHERLOCK, Specific High‐Sensitivity Enzymatic Reporter UnLOCKing; RPA, recombinase polymerase amplification; CRISPR‐MTB, CRISPR‐ Mycobacterium tuberculosis…”

“…In addition, it is worth noticing that this diagnostic tool evaluated in this study requires only 500 μL of samples and an average time of 1.5 h. Another technique was developed by Yuan et al, which is a new diagnostic platform based on distance‐associated optical characters of the AuNPs‐DNA probes (Figure 9E). 66 In this technique, universal linker ssDNA or ssRNA working as the transcleavage substrates for the Cas12a or Cas13a was applied, respectively. In addition, they then designed a pair of AuNPs‐DNA probes to hybridize to linker ssDNA or ssRNA.…”

Next-generation pathogen diagnosis with CRISPR/Cas-based detection methods

“…Importantly, the simplicity of the CRISPR/Cas-based detection system allows the rapid development of diagnostic methods for an emergency outbreak of infectious diseases. For example, integrating later flow assay (CORDS, CRISPR/Cas12a-LFD) and naked-eye detection (CRISPR/Cas-based colorimetric platform, fluorescence-based POC system), four groups quickly developed Cas12a-based on-site diagnostic assays for African swine virus (ASFV), an emergence virus for the global swine industry (Table 2) [44][45][46][47]. For the current COVID-19 pandemic [48], several research groups are developing CRISPR/Cas-based diagnostic assay [49][50][51][52][53].…”

“…Isothermal amplification that does not require instruments has been widely integrated into CRISPR-based detection [ 7 ]. For signal readout, more equipment-free methods, such as later flow assay or naked-eye view under light, should be introduced [ 41 , 45 , 46 , 55 ]. Currently, the store and delivery of the CRISPR-based detection system still need a cold chain.…”

Gold Nanoparticles for Vectorization of Nucleic Acids for Cancer Therapeutics

“…AuNPs have been characterized as efficient, specific, and selective vehicles for gene silencing and genome editing tools. CRISPR/CAS9 system is a great approach for the simultaneous edition of multiple loci, but have some limitations, such as cleavage at off-target sites [40,136]. Recently, Shahbazi et al reported an efficacious nanoformulation with low toxicity towards the delivery of CRISPR tools based on colloidal AuNPs.…”