CREST, a Cas13-Based, Rugged, Equitable, Scalable Testing (CREST) for SARS-CoV-2 Detection in Patient Samples.

The COVID-19 pandemic has taken a devastating human toll worldwide. The development of impactful guidelines and measures for controlling the COVID-19 pandemic requires continuous and widespread testing of suspected cases and their contacts through accurate, accessible, and reliable methods for SARS-CoV-2 detection. Here we describe a CRISPR-Cas13-based method for the detection of SARS-CoV-2. The assay is called CREST (Cas13-based, rugged, equitable, scalable testing), and is specific, sensitive, and highly accessible. As such, CREST may provide a low-cost and dependable alternative for SARS-CoV-2 surveillance. © 2022 Wiley Periodicals LLC. Basic Protocol: Cas13-ased detection of SARS-CoV-2 genetic material using a real-time PCR detection system Alternate Protocol: Cas13-based detection of SARS-CoV-2 genetic material using a fluorescence viewer Support Protocol 1: LwaCas13a purification Support Protocol 2: In vitro transcription of synthetic targets.

A Scalable, Easy-to-Deploy Protocol for Cas13-Based Detection of SARS-CoV-2 Genetic Material.

The COVID-19 pandemic has created massive demand for widespread, distributed tools for detecting SARS-CoV-2 genetic material. The hurdles to scalable testing include reagent and instrument accessibility, availability of highly trained personnel, and large upfront investment. Here, we showcase an orthogonal pipeline we call CREST (Cas13-based, rugged, equitable, scalable testing) that addresses some of these hurdles. Specifically, CREST pairs commonplace and reliable biochemical methods (PCR) with low-cost instrumentation, without sacrificing detection sensitivity. By taking advantage of simple fluorescence visualizers, CREST allows a binary interpretation of results. CREST may provide a point-of-care solution to increase the distribution of COVID-19 surveillance.

Comparison of Severe Acute Respiratory Syndrome Coronavirus 2 Screening Using Reverse Transcriptase-Quantitative Polymerase Chain Reaction or CRISPR-Based Assays in Asymptomatic College Students.

Importance: The reopening of colleges and universities in the US during the coronavirus disease 2019 (COVID-19) pandemic is a significant public health challenge. The development of accessible and practical approaches for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in the college population is paramount for deploying recurrent surveillance testing as an essential strategy for virus detection, containment, and mitigation. Objective: To determine the prevalence of SARS-CoV-2 in asymptomatic participants in a university community by using CREST (Cas13-based, rugged, equitable, scalable testing), a CRISPR-based test developed for accessible and large-scale viral screening. Design, Setting, and Participants: For this cohort study, a total of 1808 asymptomatic participants were screened for SARS-CoV-2 using a CRISPR-based assay and a point-of-reference reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) test. Viral prevalence in self-collected oropharyngeal swab samples collected from May 28 to June 11, 2020, and from June 23 to July 2, 2020, was evaluated. Exposures: Testing for SARS-CoV-2. Main Outcomes and Measures: SARS-CoV-2 status, viral load, and demographic information of the study participants were collected. Results: Among the 1808 participants (mean [SD] age, 27.3 [11.0] years; 955 [52.8%] female), 732 underwent testing from May to early June (mean [SD] age, 28.4 [11.7] years; 392 [53.6%] female). All test results in this cohort were negative. In contrast, 1076 participants underwent testing from late June to early July (mean [SD] age, 26.6 [10.5] years; 563 [52.3%] female), with 9 positive results by RT-qPCR. Eight of these positive samples were detected by the CRISPR-based assay and confirmed by Clinical Laboratory Improvement Amendments-certified diagnostic testing. The mean (SD) age of the positive cases was 21.7 (3.3) years; all 8 individuals self-identified as students. These metrics showed that a CRISPR-based assay was effective at capturing positive SARS-CoV-2 cases in this student population. Notably, the viral loads detected in these asymptomatic cases resemble those seen in clinical samples, highlighting the potential of covert viral transmission. The shift in viral prevalence coincided with the relaxation of stay-at-home measures. Conclusions and Relevance: These findings reveal a shift in SARS-CoV-2 prevalence in a young and asymptomatic population and uncover the leading edge of a local outbreak that coincided with rising case counts in the surrounding county and the state of California. The concordance between CRISPR-based and RT-qPCR testing suggests that CRISPR-based assays are reliable and offer alternative options for surveillance testing and detection of SARS-CoV-2 outbreaks, as is required to resume operations in higher-education institutions in the US and abroad.

The impact of SARS-CoV-2 in dementia across Latin America: A call for an urgent regional plan and coordinated response.

The SARS-CoV-2 global pandemic will disproportionately impact countries with weak economies and vulnerable populations including people with dementia. Latin American and Caribbean countries (LACs) are burdened with unstable economic development, fragile health systems, massive economic disparities, and a high prevalence of dementia. Here, we underscore the selective impact of SARS-CoV-2 on dementia among LACs, the specific strain on health systems devoted to dementia, and the subsequent effect of increasing inequalities among those with dementia in the region. Implementation of best practices for mitigation and containment faces particularly steep challenges in LACs. Based upon our consideration of these issues, we urgently call for a coordinated action plan, including the development of inexpensive mass testing and multilevel regional coordination for dementia care and related actions. Brain health diplomacy should lead to a shared and escalated response across the region, coordinating leadership, and triangulation between governments and international multilateral networks.

A Fast and Accessible Method for the Isolation of RNA, DNA, and Protein To Facilitate the Detection of SARS-CoV-2.

Management of the coronavirus disease 2019 (COVID-19) pandemic requires widespread testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A main limitation for widespread SARS-CoV-2 testing is the global shortage of essential supplies, among them RNA extraction kits. The need for commercial RNA extraction kits places a bottleneck on tests that detect SARS-CoV-2 genetic material, including PCR-based reference tests. Here, we propose an alternative method we call PEARL (precipitation-enhanced analyte retrieval) that addresses this limitation. PEARL uses a lysis solution that disrupts cell membranes and viral envelopes while simultaneously providing conditions suitable for alcohol-based precipitation of RNA, DNA, and proteins. PEARL is a fast, low-cost, and simple method that uses common laboratory reagents and offers performance comparable to that of commercial RNA extraction kits. PEARL offers an alternative method to isolate host and pathogen nucleic acids and proteins to streamline the detection of DNA and RNA viruses, including SARS-CoV-2.

The importance of naturally attenuated SARS-CoV-2in the fight against COVID-19.

The current SARS-CoV-2 pandemic is wreaking havoc throughout the world and has rapidly become a global health emergency. A central question concerning COVID-19 is why some individuals become sick and others not. Many have pointed already at variation in risk factors between individuals. However, the variable outcome of SARS-CoV-2 infections may, at least in part, be due also to differences between the viral subspecies with which individuals are infected. A more pertinent question is how we are to overcome the current pandemic. A vaccine against SARS-CoV-2 would offer significant relief, although vaccine developers have warned that design, testing and production of vaccines may take a year if not longer. Vaccines are based on a handful of different designs (i), but the earliest vaccines were based on the live, attenuated virus. As has been the case for other viruses during earlier pandemics, SARS-CoV-2 will mutate and may naturally attenuate over time (ii). What makes the current pandemic unique is that, thanks to state-of-the-art nucleic acid sequencing technologies, we can follow in detail how SARS-CoV-2 evolves while it spreads. We argue that knowledge of naturally emerging attenuated SARS-CoV-2 variants across the globe should be of key interest in our fight against the pandemic.