Light-Start CRISPR-Cas12a Reaction with Caged crRNA Enables Rapid and Sensitive Nucleic Acid Detection.

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a promising platform for nucleic acid detection. Regulating the CRISPR reaction would be extremely useful to improve the detection efficiency and speed of CRISPR diagnostic applications. Here, we have developed a light-start CRISPR-Cas12a reaction by employing caged CRISPR RNA (crRNA). When combined with recombinase polymerase amplification, a robust photocontrolled one-pot assay is achieved. The photocontrolled one-pot assay is simpler and is 50-fold more sensitive than the conventional assay. This improved detection efficiency also facilitates the development of a faster CRISPR diagnostic method. The detection of clinical samples demonstrated that 10-20 min is sufficient for effective detection, which is much faster than the current gold-standard technique PCR. We expect this advance in CRISPR diagnostics to promote its widespread detection applications in biomedicine, agriculture, and food safety.

CRISPR Technology and Its Importance in SARS-CoV-2 Treatment

Coronavirus disease-2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) predominantly affects the respiratory system. The COVID-19 pandemic has had devastating effects on the health system and the global economy worldwide. To reduce the worsening impact of the pandemic, various treatment options and vaccines have been developed. Despite these efforts the pandemic could not be stopped because of the single-stranded nature of the virus combined with the lack of proof-reading abilities of the RNA-dependent RNA polymerase (RdRp). This results in a high probability of error in the copying process and consequently, mutations occur. The increase in mutations in SARS-CoV-2 reduced the efficacy of antiviral medicines and vaccines. To fight this problem, studies were conducted on the efficacy and safety of using Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) in the diagnosis and treatment of COVID-19. Initially, discovered in archaea, CRISPR is a gene-editing tool that works by altering specific parts of the genome. In this review, we focused on the efficacy and safety of CRISPR technology in the treatment of COVID-19.Copyright © 2023 Bilimsel Tip Yayinevi. All rights reserved.

CRISPR techniques and potential for the detection and discrimination of SARS-CoV-2 variants of concern.

The continuing evolution of the SARS-CoV-2 virus has led to the emergence of many variants, including variants of concern (VOCs). CRISPR-Cas systems have been used to develop techniques for the detection of variants. These techniques have focused on the detection of variant-specific mutations in the spike protein gene of SARS-CoV-2. These sequences mostly carry single-nucleotide mutations and are difficult to differentiate using a single CRISPR-based assay. Here we discuss the specificity of the Cas9, Cas12, and Cas13 systems, important considerations of mutation sites, design of guide RNA, and recent progress in CRISPR-based assays for SARS-CoV-2 variants. Strategies for discriminating single-nucleotide mutations include optimizing the position of mismatches, modifying nucleotides in the guide RNA, and using two guide RNAs to recognize the specific mutation sequence and a conservative sequence. Further research is needed to confront challenges in the detection and differentiation of variants and sublineages of SARS-CoV-2 in clinical diagnostic and point-of-care applications.

Rapid SARS-CoV-2 Variants Enzymatic Detection (SAVED) by CRISPR-Cas12a.

The continuous and rapid surge of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with high transmissibility and evading neutralization is alarming, necessitating expeditious detection of the variants concerned. Here, we report the development of rapid SARS-CoV-2 variants enzymatic detection (SAVED) based on CRISPR-Cas12a targeting of previously crucial variants, including Alpha, Beta, Gamma, Delta, Lambda, Mu, Kappa, and currently circulating variant of concern (VOC) Omicron and its subvariants BA.1, BA.2, BA.3, BA.4, and BA.5. SAVED is inexpensive (US$3.23 per reaction) and instrument-free. SAVED results can be read out by fluorescence reader and tube visualization under UV/blue light, and it is stable for 1 h, enabling high-throughput screening and point-of-care testing. We validated SAVED performance on clinical samples with 100% specificity in all samples and 100% sensitivity for the current pandemic Omicron variant samples having a threshold cycle (CT) value of ≤34.9. We utilized chimeric CRISPR RNA (crRNA) and short crRNA (15-nucleotide [nt] to 17-nt spacer) to achieve single nucleotide polymorphism (SNP) genotyping, which is necessary for variant differentiation and is a challenge to accomplish using CRISPR-Cas12a technology. We propose a scheme that can be used for discriminating variants effortlessly and allows for modifications to incorporate newer upcoming variants as the mutation site of these variants may reappear in future variants. IMPORTANCE Rapid differentiation and detection tests that can directly identify SARS-CoV-2 variants must be developed in order to meet the demands of public health or clinical decisions. This will allow for the prompt treatment or isolation of infected people and the implementation of various quarantine measures for those exposed. We report the development of the rapid SARS-CoV-2 variants enzymatic detection (SAVED) method based on CRISPR-Cas12a that targets previously significant variants like Alpha, Beta, Gamma, Delta, Lambda, Mu, and Kappa as well as the VOC Omicron and its subvariants BA.1, BA.2, BA.3, BA.4, and BA.5 that are currently circulating. SAVED uses no sophisticated instruments and is reasonably priced ($3.23 per reaction). As the mutation location of these variations may reoccur in subsequent variants, we offer a system that can be applied for variant discrimination with ease and allows for adjustments to integrate newer incoming variants.

CRISPR/Cas-engineered technology: Innovative approach for biosensor development.

On-site and real-time clinical monitoring have been progressed dramatically by integrating biosensor science with portable digital electronic technology. Clustered regularly interspaced short palindromic repeats (CRISPR) with association of RNA-guided nucleases (CrRNA-Cas enzymes) have achieved novel CRISPR/Cas biosensing science as a promising revolutionized diagnostic technology for portable and on-site healthcare monitoring and diagnostics. Among several available CRISPR/Cas systems, CRISPR/Cas12a and CRISPR/Cas13a conjugates are utilized broadly in biosensor design, because of their capability to cleave both target and non-target sequences. With the advantages of portability, cost-effectiveness, facile operation, high durability, and reproducibility, CRISPR/Cas-based biosensing techniques are a perfect choice for designing ultra-sensitive point-of-care diagnostic devices with amplified response signals. In the present review, we summarize the advances in the CRISPR/Cas-based biosensors with the focus on healthcare and diagnostic purposes. The cooperation of nanomaterial engineering with CRISPR/Cas biosensors is also represented to attain a promising viewpoint for offering novel user-friendly test kits for announcing ultra-low levels of diverse targets in the future.

CRISPR-Cas13a cascade-based viral RNA assay for detecting SARS-CoV-2 and its mutations in clinical samples.

SARS-CoV-2 is one of the greatest threats to global human health. Point-of-care diagnostic tools for SARS-CoV-2 could facilitate rapid therapeutic intervention and mitigate transmission. In this work, we report CRISPR-Cas13a cascade-based viral RNA (Cas13C) assay for label-free and isothermal determination of SARS-CoV-2 and its mutations in clinical samples. Cas13a/crRNA was utilized to directly recognize the target of SARS-CoV-2 RNA, and the recognition events sequentially initiate the transcription amplification to produce light-up RNA aptamers for output fluorescence signal. The recognition of viral RNA via Cas13a-guide RNA ensures a high specificity to distinguish SARS-CoV-2 from MERS-CoV and SARS-CoV, as well as viral mutations. A post transcription amplification strategy was triggered after CRISPR-Cas13a recognition contributes to an amplification cascade that achieves high sensitivity for detecting SARS-CoV-2 RNA, with a limit of detection of 0.216 fM. In addition, the Cas13C assay could be able to discriminate single-nucleotide mutation, which was proven with N501Y in SARS-Cov-2 variant. This method was validated by a 100% agreement with RT-qPCR results from 12 clinical throat swab specimens. The Cas13C assay has the potential to be used as a routine nucleic acid test of SARS-CoV-2 virus in resource-limited regions.