The CRISPR/Cas System: A Customizable Toolbox for Molecular Detection.

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) are promising molecular diagnostic tools for rapidly and precisely elucidating the structure and function of genomes due to their high specificity, programmability, and multi-system compatibility in nucleic acid recognition. Multiple parameters limit the ability of a CRISPR/Cas system to detect DNA or RNA. Consequently, it must be used in conjunction with other nucleic acid amplification techniques or signal detection techniques, and the reaction components and reaction conditions should be modified and optimized to maximize the detection performance of the CRISPR/Cas system against various targets. As the field continues to develop, CRISPR/Cas systems have the potential to become an ultra-sensitive, convenient, and accurate biosensing platform for the detection of specific target sequences. The design of a molecular detection platform employing the CRISPR/Cas system is asserted on three primary strategies: (1) Performance optimization of the CRISPR/Cas system; (2) enhancement of the detection signal and its interpretation; and (3) compatibility with multiple reaction systems. This article focuses on the molecular characteristics and application value of the CRISPR/Cas system and reviews recent research progress and development direction from the perspectives of principle, performance, and method development challenges to provide a theoretical foundation for the development and application of the CRISPR/CAS system in molecular detection technology.

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-Cas assisted diagnostics: A broad application biosensing approach

In addition to its remarkable genome editing capability, the CRISPR-Cas system has proven to be very effective in many fields of application, including the biosensing of pathogenic infections, mutagenic defects, or early cancer diagnosis. Thanks to their many advantages in terms of simplicity, efficiency, and reduced time, several CRISPR-Cas systems have been described for the design of sensitive and selective analytical tools, paving the way for the development and further commercialization of next-generation diagnostics. However, CRISPR-Cas-based biosensors still need further research efforts to improve some drawbacks, such as the need for target amplification, low reproducibility, and lack of knowledge of exploited element robustness. This review aims to describe the latest trends in the design of CRISPR-Cas biosensing technologies to better highlight the insights of their advantages and to point out the limitations that still need to be overcome for their future market entry as medical diagnostics.Copyright © 2023 Elsevier B.V.

Genome editing technology and applications with the type I CRISPR system

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems, which are representative genome editing technologies, are classified into class 1 and class 2 in terms of evolutionary biology and are further classified into several subtypes. Class 2 CRISPR systems, including type II Cas9 and type V Cas12a, are the most commonly used for genome editing in eukaryotic cells, while type I CRISPR systems within Class 1 are also becoming available. Type I CRISPR recognizes longer target sequences than CRISPR-Cas9 and can induce large deletion mutations of several kilobases. These features demonstrate its potential as a novel and unique genome editing tool that can induce genetic disruption safely and reliably. Thus, it is expected to be utilized for gene therapy and industrial applications. Recently, the DNA cleavage mechanism of type I CRISPR has also revealed details from protein-complex analyses with X-ray crystallography, cryo-electron microscopy, and high-speed atomic force microscopy. The single-strand DNA trans-cleavage activity of type I CRISPR, called collateral activity, has broadened the potential application for CRISPR diagnostics, especially in the development of point-of-care testing methods for COVID-19. In this review, we present an overview of the type I CRISPR system, its application to genome editing, and genetic diagnosis using CRISPR-Cas3.Copyright © 2022

Generation of APN-chimeric gene-edited pigs by CRISPR/Cas9-mediated knock-in strategy

The prevalence of porcine enteric coronaviruses (PECs), including transmissible gastroenteritis virus (TGEV), swine acute diarrhea syndrome coronavirus (SADS-CoV), porcine delta coronavirus (PDCoV), and porcine epidemic diarrhea virus (PEDV), poses a serious threat to animal and public health. Here, we aimed to further optimize the porcine aminopeptidase N (pAPN) gene editing strategy to explore the balance between individual antiviral properties and the biological functions of pAPN in pigs. Finally, APN-chimeric gene-edited pigs were produced through a CRISPR/Cas9-mediated knock-in strategy. Further reproductive tests indicated that these gene-edited pigs exhibited normal pregnancy rates and viability. Notably, in vitro viral challenge assays further demonstrated that porcine kidney epithelial cells isolated from F1-generation gene-edited pigs could effectively inhibit TGEV infection. This study is the first to report the generation of APN-chimeric pigs, which may provide a natural host animal for characterizing PEC infection with APN and help in the development of better antiviral solutions. © 2022 Elsevier B.V.

CRISPR-Cas13d effectively targets SARS-CoV-2 variants, including Delta and Omicron, and inhibits viral infection.

The recent pandemic of variants of concern (VOC) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights the need for innovative anti-SARS-CoV-2 approaches in addition to vaccines and antiviral therapeutics. Here, we demonstrate that a CRISPR-Cas13-based strategy against SARS-CoV-2 can effectively degrade viral RNA. First, we conducted a cytological infection experiment, screened CRISPR-associated RNAs (crRNAs) targeting conserved regions of viruses, and used an in vitro system to validate functional crRNAs. Reprogrammed Cas13d effectors targeting NSP13, NSP14, and nucleocapsid transcripts achieved >99% silencing efficiency in human cells which are infected with coronavirus 2, including the emerging variants in the last 2 years, B.1, B.1.1.7 (Alpha), D614G B.1.351 (Beta), and B.1.617 (Delta). Furthermore, we conducted bioinformatics data analysis. We collected the sequence information of COVID-19 and its variants from China, and phylogenetic analysis revealed that these crRNA oligos could target almost 100% of the SARS-CoV family, including the emerging new variant, Omicron. The reprogrammed Cas13d exhibited high specificity, efficiency, and rapid deployment properties; therefore, it is promising for antiviral drug development. This system could possibly be used to protect against unexpected SARS-CoV-2 variants carrying multiple mutations.

Point-of-care CRISPR/Cas biosensing technology: A promising tool for preventing the possible COVID-19 resurgence caused by contaminated cold-chain food and packaging.

The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused great public health concern and has been a global threat due to its high transmissibility and morbidity. Although the SARS-CoV-2 transmission mainly relies on the person-to-person route through the respiratory droplets, the possible transmission through the contaminated cold-chain food and packaging to humans has raised widespread concerns. This review discussed the possibility of SARS-CoV-2 transmission via the contaminated cold-chain food and packaging by tracing the occurrence, the survival of SARS-CoV-2 in the contaminated cold-chain food and packaging, as well as the transmission and outbreaks related to the contaminated cold-chain food and packaging. Rapid, accurate, and reliable diagnostics of SARS-CoV-2 is of great importance for preventing and controlling the COVID-19 resurgence. Therefore, we summarized the recent advances on the emerging clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system-based biosensing technology that is promising and powerful for preventing the possible COVID-19 resurgence caused by the contaminated cold-chain food and packaging during the COVID-19 pandemic, including CRISPR/Cas system-based biosensors and their integration with portable devices (e.g., smartphone, lateral flow assays, microfluidic chips, and nanopores). Impressively, this review not only provided an insight on the possibility of SARS-CoV-2 transmission through the food supply chain, but also proposed the future opportunities and challenges on the development of CRISPR/Cas system-based detection methods for the diagnosis of SARS-CoV-2.

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

The CRISPR/Cas system is a protective adaptive immune system against attacks from foreign mobile genetic elements. Since the discovery of the excellent target-specific sequence recognition ability of the CRISPR/Cas system, the CRISPR/Cas system has shown excellent performance in the development of pathogen nucleic-acid-detection technology. In combination with various biosensing technologies, researchers have made many rapid, convenient, and feasible innovations in pathogen nucleic-acid-detection technology. With an in-depth understanding and development of the CRISPR/Cas system, it is no longer limited to CRISPR/Cas9, CRISPR/Cas12, and other systems that had been widely used in the past; other CRISPR/Cas families are designed for nucleic acid detection. We summarized the application of CRISPR/Cas-related technology in infectious-disease detection and its development in SARS-CoV-2 detection.

CRISPR-Cas-mediated diagnostics.

An ideal molecular diagnostic method should be sensitive, specific, low cost, rapid, portable, and easy to operate. Traditional nucleic acid detection methods based mainly on PCR technology have not only high sensitivity and specificity, but also some limitations, such as the need for expensive equipment and skilled technicians, being both time and labor intensive, and difficult to implement in some regions. However, with the continuous development of CRISPR-Cas technology and its application in molecular diagnosis, new approaches have been used for the construction of molecular diagnostic systems. In this review, we discuss recent advances in CRISPR-based molecular diagnostic technologies and highlight the revolution they bring to the field of molecular diagnostics.

CRISPR gene editing: a revolution in progress

CRISPR is a gene editing technique that has revolutionised research and has the potential to transform the treatment of many diseases. This article discusses the principles of the technique, its therapeutic applications and potential safety issues.

FNCAS9 editor linked uniform detection assay (FELUDA)

The present invention describes a method for Tusing a bacterial CRISPR Cas Ribonucleoprotein complex for detecting single nucleotide variants in RNA or DNA or more broadly, any DNA or RNA fragment, without the need for sequencing. The principle ofdiscrimination is derived from the natural property of the enzyme being used for the invention, Francisellanovicida Cas9 (FnCas9) which shows very low binding affinity to mismatched substrates. DNA is isolated either from blood, saliva, or any other biological sources like bacteria and amplified if required. For virus infected patients, samples are collected as a nasal swab and inactivated. Total RNA isolated from the sample is converted to cDNA using the reverse transcriptase enzyme. The DNA (when test material is DNA) or cDNA (when test material is RNA, like for COVID-19) is subjected to Polymerase Chain reaction, amplifying using specific primers and tagging the amplified DNA products with a ligand of choice. The detection mix consists of labelled PCR products, sgRNA-fnCAS9 complex. The detection complex can be visualized using a wide array of technologies like lateral flow, gel based cleavage assay, fluorescence based detection, in both low, medium or plate based high-throughput format. Science behind this technology will be discussed in the presentation.

MODERN MICROBIALITES and MICROBIAL MATS in VOLCANOES, WETLANDS and SALT FLATS of the CENTRAL ANDES. PROSPECTION, SCIENCE, PRESERVATION and BIOTECHNOLOGICAL APPLICATIONS

The wetlands and salt flats of the Central Andes region are unique extreme environments as they are located in high-altitude saline deserts, largely influenced by volcanic activity. Environmental factors such as ultraviolet (UV) radiation, arsenic content, high salinity, low dissolved oxygen content, extreme daily temperature fluctuation, and oligotrophic conditions, resemble the early Earth and potentially extraterrestrial conditions. The discovery of modern microbialites and microbial mats in the Central Andes during the past decade has increased the interest in this area as an early Earth analog. Along 10 years of prospection of these microbial ecosystems, we have reported, for first time for science, around 35 new systems along wetlands, lakes, volcanoes, and salt flats of Central Andes region of Argentina, Chile, and Bolivia. Microbial biodiversity and metagenomic characterization, together with ancestral biogeochemical cycles, including arsenic and carbon together with bacterial rhodopsin systems, photoreceptors characterization and plasmid biology were studied in these systems. This production of knowledge was accompanied by involvement of Andean ancestral communities, mining industries and governments in order to promote the preservation of these ancestral ecosystems. Finally, the last year, during pandemic, two stories of biotech applications based on basic knowledge of Andean extremophiles became in two Start Ups invested by the GRID X incubator program https://gridexponential.com: 1- CASPR-BIOTECH https://caspr.bio develops diagnostic kits that apply to COVID19, Hanta virus and Dengue and is based on new CRISPR-Cas systems that we discovered in the Puna salt flats and patented in the USA. 2- We founded CKAPUR https://Ckapur.com, a company that develops sustainable biotechnology applied to agriculture based on Extremophilic microorganisms: "ancestral stardust recyclers" isolated from salt flats. In this moment this Start Up is being part on Indiebio program in San Francisco USA https://indiebio.co/ In that way, studying and preserving microbial extreme biodiversity from salt pads can generate economic development in local communities through NAGOYA treaty as much as it does in mining development, only without any type of environmental impact.

CRISPR-Cas-based assays for the detection of SARS-CoV-2 RNA

The COVID-19 pandemic caused by the SARS-CoV-2 epidemic now requires the deployment of fast, sensitive and inexpensive diagnostic methods to facilitate disease management and containment worldwide. The real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay is currently the standard method for detecting SARS-CoV-2 virus. However, lack of access to laboratory resources (including rRT-PCR instrumentation, reagents, or even trained laboratory technicians) limits rRT-PCR assays from being a field-deployable and rapid diagnostic tool, especially in resource-constrained pandemic zones. Given this limitation and the necessity of expediting the diagnostic procedures, there is a strong need to develop an accurate point-of-care (POC) testing platform for COVID-19 with quick turnaround times. A POC community-level quick test (used as an initial screening test before subsequent confirmation by rRT-PCR in a central laboratory) can significantly improve diagnostic utility and ultimately reduce the burden on medical resources. CRISPR-Cas technology has now surfaced as a promising diagnostic tool with rewarding prospects of rapid detection and low cost, thereby serving as a POC one-stop platform to detect COVID 19 infection and loads. Herein, we review various hitherto CRISPR–Cas-based methodologies for the detection of SARS-CoV-2 viral RNA.

CRISPR-Cas12a-Based Detection for the Major SARS-CoV-2 Variants of Concern.

A big challenge for the control of COVID-19 pandemic is the emergence of variants of concern (VOCs) or variants of interest (VOIs) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which may be more transmissible and/or more virulent and could escape immunity obtained through infection or vaccination. A simple and rapid test for SARS-CoV-2 variants is an unmet need and is of great public health importance. In this study, we designed and analytically validated a CRISPR-Cas12a system for direct detection of SARS-CoV-2 VOCs. We further evaluated the combination of ordinary reverse transcription-PCR (RT-PCR) and CRISPR-Cas12a to improve the detection sensitivity and developed a universal system by introducing a protospacer adjacent motif (PAM) near the target mutation sites through PCR primer design to detect mutations without PAM. Our results indicated that the CRISPR-Cas12a assay could readily detect the signature spike protein mutations (K417N/T, L452R/Q, T478K, E484K/Q, N501Y, and D614G) to distinguish alpha, beta, gamma, delta, kappa, lambda, and epsilon variants of SARS-CoV-2. In addition, the open reading frame 8 (ORF8) mutations (T/C substitution at nt28144 and the corresponding change of amino acid L/S) could differentiate L and S lineages of SARS-CoV-2. The low limit of detection could reach 10 copies/reaction. Our assay successfully distinguished 4 SARS-CoV-2 strains of wild type and alpha (B.1.1.7), beta (B.1.351), and delta (B.1.617.2) variants. By testing 32 SARS-CoV-2-positive clinical samples infected with the wild type (n = 5) and alpha (n = 11), beta (n = 8), and delta variants (n = 8), the concordance between our assay and sequencing was 100%. The CRISPR-based approach is rapid and robust and can be adapted for screening the emerging mutations and immediately implemented in laboratories already performing nucleic acid amplification tests or in resource-limited settings. IMPORTANCE We described CRISPR-Cas12-based multiplex allele-specific assay for rapid SARS-CoV-2 variant genotyping. The new system has the potential to be quickly developed, continuously updated, and easily implemented for screening of SARS-CoV-2 variants in resource-limited settings. This approach can be adapted for emerging mutations and implemented in laboratories already conducting SARS-CoV-2 nucleic acid amplification tests using existing resources and extracted nucleic acid.

Manipulation of genes could inhibit SARS-CoV-2 infection that causes COVID-19 pandemics.

The year 2020 witnessed an unpredictable pandemic situation due to novel coronavirus (COVID-19) outbreaks. This condition can be more severe if the patient has comorbidities. Failure of viable treatment for such viral infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is due to lack of identification. Thus, modern and productive biotechnology-based tools are being used to manipulate target genes by introducing the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (CRISPR-associated) system. Moreover, it has now been used as a tool to inhibit viral replication. Hence, it can be hypothesized that the CRISPR/Cas system can be a viable tool to target both the SARS-CoV-2 genome with specific target RNA sequence and host factors to destroy the SARS-CoV-2 community via inhibition of viral replication and infection. Moreover, comorbidities and COVID-19 escalate the rate of mortality globally, and as a result, we have faced this pandemic. CRISPR/Cas-mediated genetic manipulation to knockdown viral sequences may be a preventive strategy against such pandemic caused by SARS-CoV-2. Furthermore, prophylactic antiviral CRISPR in human cells (PAC-MAN) along with CRISPR/Cas13d efficiently degrades the specific RNA sequence to inhibit viral replication. Therefore, we suggest that CRISPR/Cas system with PAC-MAN could be a useful tool to fight against such a global pandemic caused by SARS-CoV-2. This is an alternative preventive approach of management against the pandemic to destroy the target sequence of RNA in SARS-CoV-2 by viral inhibition.

COVID-19 Diagnostic Strategies. Part I: Nucleic Acid-Based Technologies.

The novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused respiratory infection, resulting in more than two million deaths globally and hospitalizing thousands of people by March 2021. A considerable percentage of the SARS-CoV-2 positive patients are asymptomatic or pre-symptomatic carriers, facilitating the viral spread in the community by their social activities. Hence, it is critical to have access to commercialized diagnostic tests to detect the infection in the earliest stages, monitor the disease, and follow up the patients. Various technologies have been proposed to develop more promising assays and move toward the mass production of fast, reliable, cost-effective, and portable PoC diagnostic tests for COVID-19 detection. Not only COVID-19 but also many other pathogens will be able to spread and attach to human bodies in the future. These technologies enable the fast identification of high-risk individuals during future hazards to support the public in such outbreaks. This paper provides a comprehensive review of current technologies, the progress in the development of molecular diagnostic tests, and the potential strategies to facilitate innovative developments in unprecedented pandemics.

CRISPR systems: Novel approaches for detection and combating COVID-19.

Type V and VI CRISPR enzymes are RNA-guided, DNA and RNA-targeting effectors that allow specific gene knockdown. Cas12 and Cas13 are CRISPR proteins that are efficient agents for diagnosis and combating single-stranded RNA (ssRNA) viruses. The programmability of these proteins paves the way for the detection and degradation of RNA viruses by targeting RNAs complementary to its CRISPR RNA (crRNA). Approximately two-thirds of viruses causing diseases contain ssRNA genomes. The Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has caused the outbreak of the coronavirus disease 2019 (COVID-19), which has infected more than 88 million people worldwide with near 2 million deaths since December 2019. Thus, accurate and rapid diagnostic and therapeutic tools are essential for early detection and treatment of this widespread infectious disease. For us, the CRISPR based platforms seem to be a plausible new approach for an accurate detection and treatment of SARS-CoV-2. In this review, we talk about Cas12 and Cas13 CRISPR systems and their applications in diagnosis and treatment of RNA virus mediated diseases. In continue, the SARS-CoV-2 pathogenicity, and its conventional diagnostics and antivirals will be discussed. Moreover, we highlight novel CRISPR based diagnostic platforms and therapies for COVID-19. We also discuss the challenges of diagnostic CRISPR based platforms as well as clarifying the proposed solution for high efficient selective in vivo delivery of CRISPR components into SARS-CoV-2-infected cells.