We sequenced the genomes of 5,085 SARS-CoV-2 strains causing two COVID-19 disease waves in metropolitan Houston, Texas, an ethnically diverse region with seven million residents. The genomes were from viruses recovered in the earliest recognized phase of the pandemic in Houston, and an ongoing massive second wave of infections. The virus was originally introduced into Houston many times independently. Virtually all strains in the second wave have a Gly614 amino acid replacement in the spike protein, a polymorphism that has been linked to increased transmission and infectivity. Patients infected with the Gly614 variant strains had significantly higher virus loads in the nasopharynx on initial diagnosis. We found little evidence of a significant relationship between virus genotypes and altered virulence, stressing the linkage between disease severity, underlying medical conditions, and host genetics. Some regions of the spike protein - the primary target of global vaccine efforts - are replete with amino acid replacements, perhaps indicating the action of selection. We exploited the genomic data to generate defined single amino acid replacements in the receptor binding domain of spike protein that, importantly, produced decreased recognition by the neutralizing monoclonal antibody CR30022. Our study is the first analysis of the molecular architecture of SARS-CoV-2 in two infection waves in a major metropolitan region. The findings will help us to understand the origin, composition, and trajectory of future infection waves, and the potential effect of the host immune response and therapeutic maneuvers on SARS-CoV-2 evolution.

Genome modification in budding yeast has been extremely successful largely due to its highly efficient homology-directed DNA repair machinery. Several methods for modifying the yeast genome have previously been described, many of them involving at least two-steps: insertion of a selectable marker and substitution of that marker for the intended modification. Here, we describe a CRISPR-Cas9 mediated genome editing protocol for modifying any yeast gene of interest (either essential or nonessential) in a single-step transformation without any selectable marker. In this system, the Cas9 nuclease creates a double-stranded break at the locus of choice, which is typically lethal in yeast cells regardless of the essentiality of the targeted locus due to inefficient non-homologous end-joining repair. This lethality results in efficient repair via homologous recombination using a repair template derived from PCR. In cases involving essential genes, the necessity of editing the genomic lesion with a functional allele serves as an additional layer of selection. As a motivating example, we describe the use of this strategy in the replacement of HEM2, an essential yeast gene, with its corresponding human ortholog ALAD.

Genetic variants of SARS-CoV-2 have repeatedly altered the course of the COVID-19 pandemic, and disease in individual patients. Delta variants (B.1.617.2, AY.2, and AY.3) are now the focus of international concern because they are causing widespread COVID-19 disease globally. Vaccine breakthrough cases caused by SARS-CoV-2 variants also are of considerable public health and medical concern worldwide. As part of a comprehensive project, we sequenced the genomes of 3,913 SARS-CoV-2 from patient samples acquired March 15, 2021 through July 3, 2021 in the Houston Methodist hospital system and studied vaccine breakthrough cases. During the study period Delta variants increased to cause 58% of all COVID-19 cases and spread throughout the metropolitan Houston area. In addition, Delta variants caused a significantly higher rate of vaccine breakthrough cases (19.7% compared to 5.8% for all other variants). Importantly, only 6.5% of all COVID-19 cases occurred in fully immunized individuals, and relatively few of these patients required hospitalization. Our genomic and epidemiologic data emphasize that vaccines used in the United States are highly effective in decreasing severe COVID-19 disease, hospitalizations, and deaths.

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