A Rapid Antibody Enhancement Platform in Saccharomyces cerevisiae Using an Improved, Diversifying CRISPR Base Editor.

The yeast Saccharomyces cerevisiae is commonly used to interrogate and screen protein variants and to perform directed evolution studies to develop proteins with enhanced features. While several techniques have been described that help enable the use of yeast for directed evolution, there remains a need to increase their speed and ease of use. Here we present yDBE, a yeast diversifying base editor that functions in vivo and employs a CRISPR-dCas9-directed cytidine deaminase base editor to diversify DNA in a targeted, rapid, and high-breadth manner. To develop yDBE, we enhanced the mutation rate of an initial base editor by employing improved deaminase variants and characterizing several scaffolded guide constructs. We then demonstrate the ability of the yDBE platform to improve the affinity of a displayed antibody scFv, rapidly generating diversified libraries and isolating improved binders via cell sorting. By performing high-throughput sequencing analysis of the high-activity yDBE, we show that it enables a mutation rate of 2.13 × 10-4 substitutions/bp/generation over a window of 100 bp. As yDBE functions entirely in vivo and can be easily programmed to diversify nearly any such window of DNA, we posit that it can be a powerful tool for facilitating a variety of directed evolution experiments.

RhoC Modulates Cell Junctions and Type I Interferon Response in Aggressive Breast Cancers.

Metastases are the leading cause of death in cancer patients. RhoC, a member of the Rho GTPase family, has been shown to facilitate metastasis of aggressive breast cancer cells by influencing motility, invasion, and chemokine secretion, but as yet there is no integrated model of the precise mechanism of how RhoC promotes metastasis. A common phenotypic characteristic of metastatic cells influenced by these mechanisms is dysregulation of cell-cell junctions. Thus, we set out to study how RhoA- and RhoC-GTPase influence the cell-cell junctions in aggressive breast cancers. We demonstrate that CRISPR-Cas9 knockout of RhoC in SUM 149 and MDA 231 breast cancer cells results in increased normalization of junctional integrity denoted by junction protein expression/colocalization. In functional assessments of junction stability, RhoC knockout cells have increased barrier integrity and increased cell-cell adhesion compared to wild-type cells. Whole exome RNA sequencing and targeted gene expression profiling demonstrate decreased expression of Type I interferon-stimulated genes in RhoC knockout cells compared to wild-type, and subsequent treatment with interferon-alpha resulted in significant increases in adhesion and decreases in invasiveness of wild-type cells and a dampened response to interferon-alpha stimulation with respect to adhesion and invasiveness in RhoC knockout cells. We delineate a key role of RhoC-GTPase in modulation of junctions and response to interferon, which supports inhibition of RhoC as a potential anti-invasion therapeutic strategy.

Incidence and survival of inflammatory breast cancer between 1973 and 2015 in the SEER database.

PURPOSE: Inflammatory breast cancer (IBC) is an aggressive variant characterized by erythema, edema, and "peau d'orange" of the skin progressing within 6 months. We assessed the incidence and survival of IBC in the US over four decades. METHODS: Using SEER*Stat, a case list of IBC patients diagnosed between 1973 and 2015 (n = 29,718) was extracted from SEER 18 registries by using a combination of morphology, stage, and extent of disease criteria. M1 and M0 patients were included. Age-adjusted incidence rates, relative survival rates, and mean survival time were calculated. Significance was determined as non-overlapping 95% confidence intervals. RESULTS: The overall incidence of IBC from 1973 to 2015 is 2.76 (2.73, 2.79) cases per 100,000 people, with white patients having an incidence rate of 2.63 (2.60, 2.67), black patients 4.52 (4.39, 4.65), and patients of other race 1.84 (1.76, 1.93). The overall IBC relative 5-year survival rate is 40.5% (39.0%, 42.0%), 42.5% (40.7%, 44.3%), and 29.9% (26.6%, 33.3%) for white patients and black patients, respectively. Patients diagnosed in 1978-1982 have a mean survival time of 62.3 (52.0, 72.6) months, while those diagnosed in 2008-2012 have mean survival time of 99.4 (96.4, 102.4) months. There is no significant difference in survival time between T4D patients and patients with other T staging and extent of disease coding consistent with clinical IBC presentation. CONCLUSIONS: IBC survival has increased over four decades. Despite the improvement in survival for all racial groups, a persistent survival disparity that has not narrowed over two decades remains between white and black patients.