Local monitoring of SARS-CoV-2 variants in two large California counties in 2021.

Coronavirus Disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to persist due to mutations resulting in newer, more infectious variants of concern. We aimed to leverage an ongoing private SARS-CoV-2 testing laboratory's infrastructure to monitor SARS-CoV-2 variants in two large California counties. Study enrollment was offered to adults aged 18 years or older in Los Angeles County and Riverside County who recently tested positive for SARS-CoV-2 with a polymerase chain reaction (PCR) assay. A cycle threshold value less than or equal to 30 cycles was considered a positive test for sequencing purposes. Within 5 days of study enrollment, clinician-monitored, self-collected oral fluid and anterior nares swab specimens were obtained from participants. Specimens were transported and stored at 8 °C or cooler. Samples underwent ribonucleic acid extraction, library preparation, and sequencing. SARS-CoV-2 lineages were identified using sequencing data. Participant and genomic data were analyzed using statistical tools and visualized with toolkits. The study was approved by Advarra Institutional Review Board (Pro00053729). From May 27, 2021 to September 9, 2021, 503 individuals were enrolled and underwent specimen collection. Of the 503 participants, 238 (47.3%) participants were women, 329 (63.6%) participants were vaccinated, and 221 (43.9%) participants were of Hispanic or Spanish origin. Of the cohort, 496 (98.6%) participants had symptoms at the time of collection. Among the 503 samples, 443 (88.1%) nasal specimens and 353 (70.2%) oral specimens yielded positive sequencing results. Over our study period, the prevalence of the Alpha variant of SARS-CoV-2 decreased (initially 23.1% [95% confidence interval (95% CI): 0-0.49%] to 0% [95% CI 0.0-0.0%]) as the prevalence of the Delta variant of SARS-CoV-2 increased (initially 33.3% [95% CI 0.0-100.0%] to 100.0% [95% CI 100.0-100.0%]). A strain that carried mutations of both Delta and Mu was identified. We found that outpatient SARS-CoV-2 variant surveillance could be conducted in a timely and accurate manner. The prevalence of different variants changed over time. A higher proportion of nasal specimens yielded results versus oral specimens. Timely and regional outpatient SARS-CoV-2 variant surveillance could be used for public health efforts to identify changes in SARS-CoV-2 strain epidemiology.

Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacterial and Mammalian Cells via Dynamic RNA Nanotechnology.

A guide RNA (gRNA) directs the function of a CRISPR protein effector to a target gene of choice, providing a versatile programmable platform for engineering diverse modes of synthetic regulation (edit, silence, induce, bind). However, the fact that gRNAs are constitutively active places limitations on the ability to confine gRNA activity to a desired location and time. To achieve programmable control over the scope of gRNA activity, here we apply principles from dynamic RNA nanotechnology to engineer conditional guide RNAs (cgRNAs) whose activity is dependent on the presence or absence of an RNA trigger. These cgRNAs are programmable at two levels, with the trigger-binding sequence controlling the scope of the effector activity and the target-binding sequence determining the subject of the effector activity. We demonstrate molecular mechanisms for both constitutively active cgRNAs that are conditionally inactivated by an RNA trigger (ON → OFF logic) and constitutively inactive cgRNAs that are conditionally activated by an RNA trigger (OFF → ON logic). For each mechanism, automated sequence design is performed using the reaction pathway designer within NUPACK to design an orthogonal library of three cgRNAs that respond to different RNA triggers. In E. coli expressing cgRNAs, triggers, and silencing dCas9 as the protein effector, we observe a median conditional response of ≈4-fold for an ON → OFF "terminator switch" mechanism, ≈15-fold for an ON → OFF "splinted switch" mechanism, and ≈3-fold for an OFF → ON "toehold switch" mechanism; the median crosstalk within each cgRNA/trigger library is <2%, ≈2%, and ≈20% for the three mechanisms. To test the portability of cgRNA mechanisms prototyped in bacteria to mammalian cells, as well as to test generalizability to different effector functions, we implemented the terminator switch in HEK 293T cells expressing inducing dCas9 as the protein effector, observing a median ON → OFF conditional response of ≈4-fold with median crosstalk of ≈30% for three orthogonal cgRNA/trigger pairs. By providing programmable control over both the scope and target of protein effector function, cgRNA regulators offer a promising platform for synthetic biology.

Affinity flow fractionation of cells via transient interactions with asymmetric molecular patterns.

Flow fractionation of cells using physical fields to achieve lateral displacement finds wide applications, but its extension to surface molecule-specific separation requires labeling. Here we demonstrate affinity flow fractionation (AFF) where weak, short-range interactions with asymmetric molecular patterns laterally displace cells in a continuous, label-free process. We show that AFF can directly draw neutrophils out of a continuously flowing stream of blood with an unprecedented 400,000-fold depletion of red blood cells, with the sorted cells being highly viable, unactivated, and functionally intact. The lack of background erythrocytes enabled the use of AFF for direct enumeration of neutrophils by a downstream detector, which could distinguish the activation state of neutrophils in blood. The compatibility of AFF with capillary microfluidics and its ability to directly separate cells with high purity and minimal sample preparation will facilitate the design of simple and portable devices for point-of-care diagnostics and quick, cost-effective laboratory analysis.

Effects of ligands with different water solubilities on self-assembly and properties of targeted nanoparticles.

The engineering of drug-encapsulated targeted nanoparticles (NPs) has the potential to revolutionize drug therapy. A major challenge for the smooth translation of targeted NPs to the clinic has been developing methods for the prediction and optimization of the NP surface composition, especially when targeting ligands (TL) of different chemical properties are involved in the NP self-assembly process. Here we investigated the self-assembly and properties of two different targeted NPs decorated with two widely used TLs that have different water solubilities, and developed methods to characterize and optimize NP surface composition. We synthesized two different biofunctional polymers composed of poly(lactide-co-glycolide)-b-polyethyleneglycol-RGD (PLGA-PEG-RGD, high water solubility TL) and PLGA-PEG-Folate (low water solubility TL). Targeted NPs with different ligand densities were prepared by mixing TL-conjugated polymers with non-conjugated PLGA-PEG at different ratios through nanoprecipitation. The NP surface composition was quantified and the results revealed two distinct nanoparticle assembly behaviors: for the case of PLGA-PEG-RGD, nearly all RGD molecules conjugated to the polymer were found to be on the surface of the NPs. In contrast, only ∼20% of the folate from PLGA-PEG-Folate was present on the NP surface while the rest remained presumably buried in the PLGA NP core due to hydrophobic interactions of PLGA and folate. Finally, in vitro phagocytosis and cell targeting of NPs were investigated, from which a window of NP formulations exhibiting minimum uptake by macrophages and maximum uptake by targeted cells was determined. These results underscore the impact that the ligand chemical properties have on the targeting capabilities of self-assembled targeted nanoparticles and provide an engineering strategy for improving their targeting specificity.