COVID-19 Variant Detection with a High-Fidelity CRISPR-Cas12 Enzyme.

Laboratory tests for the accurate and rapid identification of SARS-CoV-2 variants can potentially guide the treatment of COVID-19 patients and inform infection control and public health surveillance efforts. Here, we present the development and validation of a rapid COVID-19 variant DETECTR assay incorporating loop-mediated isothermal amplification (LAMP) followed by CRISPR-Cas12 based identification of single nucleotide polymorphism (SNP) mutations in the SARS-CoV-2 spike (S) gene. This assay targets the L452R, E484K/Q/A, and N501Y mutations, at least one of which is found in nearly all major variants. In a comparison of three different Cas12 enzymes, only the newly identified enzyme CasDx1 was able to accurately identify all targeted SNP mutations. An analysis pipeline for CRISPR-based SNP identification from 261 clinical samples yielded a SNP concordance of 97.3% and agreement of 98.9% (258 of 261) for SARS-CoV-2 lineage classification, using SARS-CoV-2 whole-genome sequencing and/or real-time RT-PCR as test comparators. We also showed that detection of the single E484A mutation was necessary and sufficient to accurately identify Omicron from other major circulating variants in patient samples. These findings demonstrate the utility of CRISPR-based DETECTR as a faster and simpler diagnostic method compared with sequencing for SARS-CoV-2 variant identification in clinical and public health laboratories.

Rapid Detection of 2019 Novel Coronavirus SARS-CoV-2 Using a CRISPR-based DETECTR Lateral Flow Assay.

An outbreak of novel betacoronavirus, SARS-CoV-2 (formerly named 2019-nCoV), began in Wuhan, China in December 2019 and the COVID-19 disease associated with infection has since spread rapidly to multiple countries. Here we report the development of SARS-CoV-2 DETECTR, a rapid (~30 min), low-cost, and accurate CRISPR-Cas12 based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA extracts. We validated this method using contrived reference samples and clinical samples from infected US patients and demonstrated comparable performance to the US CDC SARS-CoV-2 real-time RT-PCR assay.

CRISPR-Cas12-based detection of SARS-CoV-2.

An outbreak of betacoronavirus severe acute respiratory syndrome (SARS)-CoV-2 began in Wuhan, China in December 2019. COVID-19, the disease associated with SARS-CoV-2 infection, rapidly spread to produce a global pandemic. We report development of a rapid (<40 min), easy-to-implement and accurate CRISPR-Cas12-based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA extracts. We validated our method using contrived reference samples and clinical samples from patients in the United States, including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections. Our CRISPR-based DETECTR assay provides a visual and faster alternative to the US Centers for Disease Control and Prevention SARS-CoV-2 real-time RT-PCR assay, with 95% positive predictive agreement and 100% negative predictive agreement.

Telomere length measurement as a clinical biomarker of aging and disease.

Telomere length measurement is increasingly recognized as a clinical gauge for age-related disease risk. There are several methods for studying blood telomere length (BTL) as a clinical biomarker. The first is an observational study approach, which directly measures telomere lengths using either cross-sectional or longitudinal patient cohorts and compares them to a population of age- and sex-matched individuals. These direct traceable measurements can be considered reflective of an individual's current health or disease state. Escalating interest in personalized medicine, access to high-throughput genotyping and resulting acquisition of large volumes of genetic data corroborates the second method, Mendelian randomization (MR). MR employs telomere length-associated genetic variants to indicate predisposition to disease risk based on the genomic composition of the individual. When assessed from cells in the bloodstream, telomeres can show variation from their genetically predisposed lengths due to environmental-induced changes. These alterations in telomere length act as an indicator of cellular health, which, in turn, can provide disease risk status. Overall, BTL measurement is a dynamic marker of biological health and well-being that together with genetically defined telomere lengths can provide insights into improved healthcare for the individual.

Analytical Validation of Relative Average Telomere Length Measurement in a Clinical Laboratory Environment.

BACKGROUND: Average telomere length in whole blood has become a biomarker of aging, disease, and mortality risk across a broad range of clinical conditions. The most common method of telomere length measurement for large patient sample sets is based on quantitative PCR (qPCR). For laboratory-developed tests to be performed on clinical samples, they must undergo a rigorous analytical validation, currently regulated under CLIA. METHODS: Whole blood samples from 40 donors were used in the analytical validation of methods for relative average telomere length (rATL) measurement. Three technical replicate DNA samples were extracted from each whole blood sample and placed in three independent wells on a sample plate. Each of these sample plates was assayed 12 times during the validation process. The study was conducted over a 20-day period, once in the morning and once in the evening, using 3 different operators. RESULTS: Our process of rATL measurement beginning with DNA extraction followed by qPCR-based assay resulted in repeatability and reproducibility CV of <5% and amplification efficiencies near 100%. The validated assay was used to establish a reference interval derived from 2 cohorts of individuals: (a) San Francisco Bay area (n = 504) and (b) a US cross-sectional, demographic population (n = 357). CONCLUSIONS: We present advances in the establishment of a highly reproducible analytically validated process for determining rATLs in a CLIA laboratory environment.

Top3-Rmi1 dissolve Rad51-mediated D loops by a topoisomerase-based mechanism.

The displacement loop (D loop) is a DNA strand invasion product formed during homologous recombination. Disruption of nascent D loops prevents recombination, and during synthesis-dependent strand annealing (SDSA), disruption of D loops extended by DNA polymerase ensures a non-crossover outcome. The proteins implicated in D loop disruption are DNA motor proteins/helicases that act by moving DNA junctions. Here we report that D loops can also be disrupted by DNA topoisomerase 3 (Top3), and this disruption depends on Top3's catalytic activity. Yeast Top3 specifically disrupts D loops mediated by yeast Rad51/Rad54; protein-free D loops or D loop mediated by bacterial RecA protein or human RAD51/RAD54 resist dissolution. Also, the human Topoisomerase IIIa-RMI1-RMI2 complex is capable of dissolving D loops. Consistent with genetic data, we suggest that the extreme growth defect and hyper-recombination phenotype of Top3-deficient yeast cells is partially a result of unprocessed D loops.

DNA damage induces alternative lengthening of telomeres (ALT) associated promyelocytic leukemia bodies that preferentially associate with linear telomeric DNA.

The linear chromosomes of vertebrates terminate in telomeres that consist of a tandemly repeated hexameric sequence, 5'TTAGGG3'. Telomeres form a protective loop structure (t-loop), which is thought to prevent them from being recognized as a double-strand break. Approximately 10% of human tumors prevent shortening of their telomeres by using a recombination-mediated alternative lengthening of telomeres (ALT) mechanism. ALT-positive human cells contain extrachromosomal telomere repeat (ECTR) DNA that may either be circular or linear. It has been proposed that ECTR may be generated by recombination events involving the t-loop. A proportion of the cells within ALT-positive cell populations contain promyelocytic leukemia (PML) nuclear bodies that contain telomeric DNA and telomere-binding proteins that are called ALT-associated PML bodies (APB). Although the presence of APBs is very useful for determining whether tumors and cell lines use the ALT mechanism, the function of APBs is unknown. It has previously been shown that telomeric DNA is particularly susceptible to damage by hydrogen peroxide and N-methyl-N'-nitro-N-nitrosoguanidine. We report here that these DNA-damaging agents induce both linear and circular ECTR DNA in ALT cells and increase the proportion of cells that contain APBs. We partially purified APBs and showed that the telomeric repeat DNA they contain is predominantly linear. We propose that a function of APBs is to sequester linear telomeric DNA.

Telomerase-independent telomere length maintenance in the absence of alternative lengthening of telomeres-associated promyelocytic leukemia bodies.

Immortal tumor cells and cell lines employ a telomere maintenance mechanism that allows them to escape the normal limits on proliferative potential. In the absence of telomerase, telomere length may be maintained by an alternative lengthening of telomeres (ALT) mechanism. All human ALT cell lines described thus far have nuclear domains of unknown function, termed ALT-associated promyelocytic leukemia bodies (APB), containing promyelocytic leukemia protein, telomeric DNA and telomere binding proteins. Here we describe telomerase-negative human cells with telomeres that contain a substantial proportion of nontelomeric DNA sequences (like telomerase-null Saccharomyces cerevisiae survivor type I cells) and that are maintained in the absence of APBs. In other respects, they resemble typical ALT cell lines: the telomeres are highly heterogeneous in length (ranging from very short to very long) and undergo rapid changes in length. In addition, these cells are capable of copying a targeted DNA tag from one telomere into other telomeres. These data show that APBs are not always essential for ALT-mediated telomere maintenance.

Cys(9), Cys(104) and Cys(207) of simian virus 40 Vp1 are essential for infectious virion formation in CV-1 cells.

Structural studies have implicated Cys(9), Cys(104) and Cys(207) of simian virus 40 (SV40) Vp1 in disulfide bond formation. Recently, we have shown the three cysteines to be essential for disulfide linkage of Vp1 complexes in vitro. Here, the role of the three cysteines was explored during the course of SV40 infection. Single-, double- and triple-mutant Vp1 at Cys(9), Cys(104) and Cys(207) continued to localize to the nuclei of transfected CV-1 cells and to bind DNA, but showed a range of abilities to form plaques. Only mutants containing the Cys(9)-->Ser change showed defects in plaque formation. Single mutants at Cys(9) formed small plaques; mutants at Cys(9). Cys(104), Cys(9). Cys(207) and Cys(9). Cys(104). Cys(207) formed no plaques. All three isolated revertants contained back-mutations at the Vp1 Cys(9) codon. These results further confirm the involvement of the three Vp1 cysteines in protein-protein interactions during virus assembly. Cys(9) is critical for production of wild-type infectious virions, whereas Cys(104) and Cys(207) play secondary roles.