Rapid detection of HPV16/18 based on a CRISPR-Cas13a/Cas12a dual-channel system

Abstract

Cervical cancer is the leading cause of cancer deaths in women in the developing world, with High-risk HPV16 and HPV18 accounting for approximately 70% of all cervical cancers. Early detection...

Development of a Naked Eye CRISPR-Cas12a and -Cas13a Multiplex Point-of-Care Detection of Genetically Modified Swine

“…Compared to earlier CRISPR nucleic acid assays, this multiplex CRISPR nucleic acid assay mediated by the CRISPR-Cas12a and -Cas13a systems can simultaneously detect two target genes in a single tube reaction, reducing the cost of amplification and the number of tubes required, effectively reducing amplicon nucleic acid contamination from the environment. − The outstanding advantage of this multiplex CRISPR nucleic acid assay compared to other CRISPR colorimetric/fluorescence assays is the use of modified green-red-yellow, fluorescent signal conversion ssDNA-FQ and ssRNA-FQ reporters combinations suitable for visualization with the naked eye, eliminating the need for expensive microplate readers and specialized hand-held devices for visualization. − …”

“…The CRISPR-Cas12a and -Cas13a-based dual-channel system combined with multiplex RPA or recombinase-aided amplification (RAA) can rapidly detect nucleic acids. However, this approach relies on costly dual-channel fluorescence unsuitable for large-scale field applications. , We previously developed ssDNA-FQ reporters with JOE and ROX fluorophores to increase the fluorescent signal for easy visualization and differentiation during nucleic acid detection . Moreover, we evaluated the performance of 16 types of ssDNA-FQ reporters for use with CRISPR/Cas12a-based visual colorimetric and fluorescent assays .…”

Advanced technologies towards improved HPV diagnostics

“…Currently, there is a plethora of different caspases, for example, Cas9, Cas12, Cas13, Cas14, and their subtypes, 106,107 that specifically cleave various target molecules such as single‐stranded (ss) DNA, double‐stranded (ds) DNA, ss RNA with cis‐ and/or trans‐cleavage activity, 108 or even unwind dsDNA 109 . Accordingly, CRISPR‐Cas system has been employed in detection of wide range of biomarkers, including microRNAs, 110,111 DNA methylation, 112 single point mutations, 113,114 various bacteria such as Salmonella , 115,116 Yersinia pestis , 117 or viruses such as SARS‐CoV‐2, 118 EBV, 119 as well as HPV 120–142 . Interestingly, CRISPR‐Cas technology was recently used also in a treatment of HPV infection‐associated cervical cancer.…”

“…Although majority of CRISPR-based studies use some DNA amplification technique before detection, [122][123][124][125][126][127][129][130][131]133,[137][138][139][140][141][142] several works reported also amplification-free strategies. 121,128,132,134,146 An interesting technology called polydisperse droplet digital CRISPR-Cas-based assay has been introduced by Xue et al 134 Results from above studies using CRISPR-Cas system implies that it offers good sequence resolution at a single nucleotide level, but discrimination of individual genotypes would require very precise design of gRNA.…”

“…Although majority of CRISPR‐based studies use some DNA amplification technique before detection, 122–127,129–131,133,137–142 several works reported also amplification‐free strategies 121,128,132,134,146 . An interesting technology called polydisperse droplet digital CRISPR‐Cas‐based assay has been introduced by Xue et al 134 whereby the reaction mixture containing CRISPR‐Cas components and a fluorescent dye‐labeled reporter probe was vortexed with oil to generate multiple droplets.…”