CRISPR-Cas12a assisted specific detection of mpox virus.

Mpox virus, a member of genus Orthopoxvirus, causes rash and flu-like symptoms in humans. In the recent global outbreak, it was reported from several geographical areas that have not historically reported mpox. Point of care, sensitive and specific mpox diagnostic assays are critical in checking the spread of the disease. We have developed a clustered regularly interspaced short palindromic repeats associated Cas12a nuclease-based assay for detecting mpox virus. Mpox specific conserved sequences were identified in polA (E9L) gene which differ by a single nucleotide polymorphism (SNP) from all the viruses present in the genus Orthopoxvirus. This SNP was exploited in our assay to specifically distinguish mpox virus from other related orthopox viruses with a limit of detection of 1 copy/µl in 30 min. The assay exhibits a sensitive and specific detection of mpox virus which can prove to be of practical value for its surveillance in areas infected with multiple orthopox viruses, especially in hotspots of mpox virus infections.

An improved, simple and field-deployable CRISPR-Cas12a assay for the detection of SARS-CoV-2.

AIMS: The RT-PCR is the most popular confirmatory test for SARS-CoV-2. It is sensitive, but high instrumentation cost makes it difficult for use outside routine clinical setup. This has necessitated the development of alternative methods such as CRISPR-based DETECTR method which uses lateral flow technology. Although accurate and sensitive, this method is limited by complex steps and recurrent cost of high-quality lateral flow strips. The main goal of this study was to improve the Cas12a-based SARS-CoV-2 DETECTR method and develop a portable and field-deployable system to reduce the recurring consumable cost. METHODS AND RESULTS: Specific regions of N and E genes from SARS-CoV-2 virus and human RNase P (internal control) were reverse transcribed (RT) and amplified by loop-mediated isothermal amplification (LAMP). The amplified products were detected by a Cas12a-based trans-cleavage reaction that generated a fluorescent signal which could be easily visualized by naked eye. Detection of internal control, RNase P gene was improved and optimized by redesigning RT-LAMP primers. A number of steps were reduced by combining the reagents related to the detection of Cas12a trans-cleavage reaction into a single ready-to-use mix. A portable, cost-effective battery-operated instrument, CRISPR-CUBE was developed to run the assay and visualize the outcome. The method and instrument were validated using both contrived and patient samples. CONCLUSIONS: The simplified CRISPR-based SARS-CoV-2 detection and instrument developed in this study, along with improved design for internal control detection allows for easier, more definitive viral detection requiring only reagents, consumables and the battery operable CRISPR-CUBE. SIGNIFICANCE AND IMPACT OF STUDY: Significant improvement in Cas12 method, coupled with simple visualization of end point makes the method and instrument deployable at the point-of-care (POC) for SARS-CoV-2 detection, without any recurrent cost for the lateral flow strips which is used in other POC methods.

Type I-E CRISPR-Cas System as a Defense System in Saccharomyces cerevisiae.

Defense against viruses and other mobile genetic elements (MGEs) is important in many organisms. The CRISPR-Cas systems found in bacteria and archaea constitute adaptive immune systems that can acquire the ability to target previously unrecognized MGEs. No CRISPR-Cas system is found to occur naturally in eukaryotic cells, but here, we demonstrate interference by a type I-E CRISPR-Cas system from Escherichia coli introduced in Saccharomyces cerevisiae. The designed CRISPR arrays are expressed and processed properly in S. cerevisiae. Targeted plasmids display reduced transformation efficiency, indicative of DNA cleavage. IMPORTANCE Genetic inactivation of viruses and other MGEs is an important tool with application in both research and therapy. Gene editing using, e.g., Cas9-based systems, can be used to inactivate MGEs in eukaryotes by introducing specific mutations. However, type I-E systems processively degrade the target which allows for inactivation without detailed knowledge of gene function. A reconstituted CRISPR-Cas system in S. cerevisiae can also function as a basic research platform for testing the role of various factors in the interference process.

The era of Cas12 and Cas13 CRISPR-based disease diagnosis.

Clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein (Cas) systems, since their discovery, have found growing applications in cell imaging, transcription modulation, therapeutics and diagnostics. Discovery of Cas12 and Cas13 have brought a new dimension to the field of disease diagnosis. These endonucleases have been extensively used for diagnosis of viral diseases in humans and animals and to a lesser extent in plants. The exigency of SARS-CoV-2 pandemic has highlighted the potential of CRISPR-Cas systems and sparked the development of innovative point-of-care diagnostic technologies. Rapid adaptation of CRISPR-chemistry combined with sensitive read-outs for emerging pathogens make them ideal candidates for detection and management of diseases in future. CRISPR-based approaches have been recruited for the challenging task of cancer detection and prognosis. It stands to reason that the field of CRISPR-Cas-based diagnosis is likely to expand with Cas12 and Cas13 playing a pivotal role. Here we focus exclusively on Cas12- and Cas13-based molecular diagnosis in humans, animals and plants including the detection of SARS-coronavirus. The CRISPR-based diagnosis of plant and animal diseases have not found adequate mention in previous reviews. We discuss various advancements, the potential shortfalls and challenges in the widespread adaptation of this technology for disease diagnosis.

DNA repair pathways important for the survival of Escherichia coli to hydrogen peroxide mediated killing.

Escherichia coli exposed to 1-3 mM hydrogen peroxide undergo killing which is designated as the mode-one killing which is a result of oxidative DNA damage. Oxidative stress mediated DNA damage can be repaired by various DNA repair pathways like base excision repair, nucleotide excision repair and homologous recombination repair. In this study we have investigated the role of multiple DNA repair pathways in survival to oxidative killing and assessed their relative importance. Results show that both nucleotide excision repair pathway as well as the RecF pathway of recombination repair are important for repair of the DNA damage caused by exposure to hydrogen peroxide. The study also provides the evidence that RecG helicase which is known for the resolution of Holliday junction intermediates plays a critical role in the survival of mode-one killing by peroxide. There is a severe impact on the survival of repair mutants when parameters like aeration and growth medium are changed. Low aeration and growth in minimal medium provide significant protection from the mode-one killing suggesting that under natural conditions Escherichia coli cells are likely to be protected from the oxidative stress mediated DNA damage.

CRISPR-Cas-Mediated Gene Silencing Reveals RacR To Be a Negative Regulator of YdaS and YdaT Toxins in Escherichia coli K-12.

Bacterial genomes are rich in horizontally acquired prophages. racR is an essential gene located in the rac prophage that is resident in many Escherichia coli genomes. Employing a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-based gene silencing approach, we show that RacR is a negative regulator of the divergently transcribed and adjacent ydaS-ydaT operon in Escherichia coli K-12. Overexpression of YdaS and YdaT due to RacR depletion leads to cell division defects and decrease in survival. We further show that both YdaS and YdaT can act independently as toxins and that RacR serves to counteract the toxicity by tightly downregulating the expression of these toxins. IMPORTANCEracR is an essential gene and one of the many poorly studied genes found on the rac prophage element that is present in many Escherichia coli genomes. Employing a CRISPR-based approach, we have silenced racR expression to various levels and elucidated its physiological consequences. We show that the downregulation of racR leads to upregulation of the adjacent ydaS-ydaT operon. Both YdaS and YdaT act as toxins by perturbing the cell division resulting in enhanced cell killing. This work establishes a physiological role for RacR, which is to keep the toxic effects of YdaS and YdaT in check and promote cell survival. We, thus, provide a rationale for the essentiality of racR in Escherichia coli K-12 strains.