Application of CRISPR/Cas Systems in the Nucleic Acid Detection of Infectious Diseases

Figure 1. Overview of CRISPR-Cas enzyme activities and their catalytic mechanisms (A) Cas9 can cleave the target and non-target strands of DNA; a short trinucleotide PAM is also essential for the initial DNA binding; (B) Cas12a can cleave dsDNA under the guidance of gRNA. The Cas12a enzyme recognizes the PAM of the original T-rich spacer and then recognizes the target sequence to generate PAM distal dsDNA breaks with staggered 5′ and 3′ ends, and Cas12 has the side chains trans-cleavage activity. At the time that the sgRNA-guided DNA is combined in Cas12, Cas12 will release a powerful, indiscriminate single-stranded DNA (ssDNA) cleavage activity; (C): Cas13 can activate its single-stranded RNA (ssRNA) cleavage activity by binding to crRNA, and it has a additional cleavage activity triggered by the target RNA; (D) Cas14 protein is a RNA-guided nuclease and can recognize the target ssDNA without restriction sequences and cleave it, and also can non-specifically cleave the surrounding ssDNA nucleases molecule; (E) Cas10 is a multi-component and multi-pronged immune effector that can be activated by viral RNA, and the viral RNA will activate Cas10 polymerization enzymes that produce about 1000…

Figure 2. Applications of CRISPR/Cas9 technology: (A) NASBACC designed a sgRNA based on the NGG PAM, a specific sensor trigger sequence was added on the amplified product after NASBA amplified, if there is a target sequence, the added sensor sequence will be cut off, and subsequent responses cannot be triggered; otherwise, the added sensor sequence will trigger the sensor response; (B) Cas-EXPAR can specifically cleave the target sequence under the guide of the designed sgRNA, produce cleaved fragments (X), X hybridizes to the EXPAR template and is extended along the template by DNA polymerase from its 3′ end. The subsequently formed duplex is cleaved by Nease; a copy of X is released from the template, finally combined with fluorescence intensity analysis to achieve detection; (C): UiO66-platform-based CRISPR/Cas9 designed two Cas9/sgRNA complexes that recognize and cleave the target DNA to produce short ssDNA and perform rolling circle amplification; when long ssDNA is present, the fluorescent probe will dissociate from UiO66 and combine it with long ssDNA to regenerate the fluorescent signal; finally, the changed fluorescence intensity is detected to detect the presence of the target…

Figure 3. Applications of CRISPR/Cas9 technology: (A) PC Reporter has two paired dCas9/sgRNA complexes that are respectively connected to the N-terminal and C-terminal of the firefly luciferase (NFluc and CFluc); when the adjacent sequences are detected in sample, the complementary upstream and downstream segments combined and generated luminescence. (B) Vigilant designed a reporting ssDNA with the 5′-end of the 25-BP VirD2 recognition sequence and the 3′-end of the biotinylated sequence; when the target nucleic acid sequence is present, biotin-reporting ssDNA-VirD2-Cas9-sgRNA-targeting ssDNA complexes are formed, and finally the results will be reported by IFA; (C): FLASH-NGS, block the sample genomic DNA or cDNA with phosphatase at first and then combined the recombinant Cas9 and multiple-guide RNAs to cleave the sequence of interest into Illumina sequencing-size fragments; through subsequent amplification, the target sequence is enriched in the background and combined with the sequencing flow cell to achieve multiple detection.

Figure 4. Applications of CRISPR/Cas12technology: (A) RAA-based E-CRISPR, uses MB to modify the ssDNA reporter gene and assemble it on the working electrode, the sample is first amplified by RAA, when the target sequence exists, non-specifically cleaves the MB-modified reporter gene on the electrode surface, finally analyzed by SWV to measure the microelectrochemical signal before and after the introduction of the target nucleic acid sequence; (B) EIS-CRISPR, fixes ssDNA on a gold electrode to limit the electronic communication between the electrode and the solution; when the target DNA exists, the Cas12/gRNA system binds to the target DNA and trans-cleaves the ssDNA on the gold electrode and accelerates the electron transfer between the electrode and the solution, detecting subtle changes in the electrode surface current at last.

Figure 5. Applications of CRISPR/Cas13 and CRISPR/Cas14 technology: (A) LLPS-CRISPR, combined with the collateral cleavage activity of Cas12a/Cas13a, cleaves long-chain into short-chain nucleotides when the target sequence is present; then the solution will become clear afterwards; (B) Light-up aptamer-based-Cas13a introduces a new light-up RNA aptamer broccoli/DFHBI-1T complex; when the target sequence is present, Cas13a digests the aptamer broccoli, and the high-fluorescence bound-state DFHBI-1T becomes the low-fluorescence free state; (C) APC-Cas’s aptamer domain will specifically recognize and bind to the target pathogen, so that AP expands from a hairpin-like inactive structure and transforms into an active structure; the primer domain can be combined with the primer, and then, with the participation of DNA polymerase, AP is used as the template chain to generate dsDNA, which replaces the target pathogen and realizes the first amplification; then the T7 promoter domain is amplified by T7 RNA polymerase to achieve the second step of amplification; subsequently, the Cas13a/crRNA complex recognizes the ssRNA produced by the second step and non-specifically cleaves a large number of…

Figure 6. Applications of CRISPR/Cas14 technology: TSPE-Cas14a, designed a tag-specific primer containing two domains (primer sequence domain matching the target, and a tag sequence domain matching sgRNA); after the target sequence is matched, the primer sequence domain begins to be amplified and enriched; then the target nucleic acid is separated from the mixture using streptavidin-coated magnetic beads, and finally, matches sgRNA identifying the tag sequence domain and cleaves the fluorescence quenching reporter gene, resulting in an enhanced fluorescence signal to achieve detection.

Figure 7. Applications of CRISPR/Cas10 technology: (A) MORIARTY, when the target sequence is present, activates Csm1 and synthesizes cOA6 with the participation of divalent ions and ATP, and then cOA6 activates the RNase activity of Csm6, lastly cleaving RNA-FAM; (B) SCOPE designed a TtCmr/crRNA complex targeting RNA; after the complex recognizes the target RNA, it can produce cOA molecules, which in turn triggers the cleavage of the reporter RNA by TTHB144, thereby generating a detectable fluorescent signal for result reading.