CRISPR/Cas12a-mediated CHO genome engineering can be effectively integrated at multiple stages of the cell line generation process for bioproduction.

Most biopharmaceuticals produced today are generated using Chinese hamster ovary (CHO) cells, therefore significant attention is focused on methods to improve CHO cell productivity and product quality. The discovery of gene-editing tools, such as CRISPR/Cas9, offers new opportunities to improve CHO cell bioproduction through cell line engineering. Recently an additional CRISPR-associated protein, Cas12a (Cpf1), was shown to be effective for gene editing in eukaryotic cells, including CHO. In this study, we demonstrate the successful application of CRISPR/Cas12a for the generation of clonally derived CHO knockout (KO) cell lines with improved product quality attributes. While we found Cas12a efficiency to be highly dependent on the targeting RNA used, we were able to generate CHO KO cell lines using small screens of only 96-320 clonally derived cell lines. Additionally, we present a novel bulk culture analysis approach that can be used to quickly assess CRISPR RNA efficiency and determine ideal screen sizes for generating genetic KO cell lines. Most critically, we find that Cas12a can be directly integrated into the cell line generation process through cotransfection with no negative impact on titer or screen size. Overall, our results show CRISPR/Cas12a to be an efficient and effective CHO genome editing tool.

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae.

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.

An analysis of inner-city students' attitudes towards violence before and after participation in the "Cradle to Grave" programme.

BACKGROUND: Cradle to Grave (C2G), a hospital-based violence prevention programme, brings inner-city youth into an urban Level I trauma centre to follow the path of an adolescent gunshot victim from trauma bay to morgue. We hypothesised that C2G alters student attitudes towards gun violence. METHODS: Eighty-eight adolescents were prospectively enrolled. With parental and student consent, students completed the Attitudes Towards Guns and Violence Questionnaire (AGVQ), a previously validated and reliable social science assessment tool. Two weeks later, the students participated in C2G. The survey was re-administered four weeks after C2G participation. AGVQ results are reported both as a total score and as a breakdown of the four component subscales. Higher AGVQ scores indicate proclivity towards violence. ANOVA compared scores with respect to demographics and type of school (public vs. charter). RESULTS: C2G altered student's attitudes towards guns and violence. Of 43 public school students, total scores decreased following C2G (p=0.02). The greatest attitudinal change occurred in subscale 1, "Aggressive Response to Shame" (p<0.01). C2G failed to produce significant changes AGVQ scores in the 45 students attending a city charter school. The two groups were found to have baseline differences, with public school students showing higher baseline tendencies towards violence. CONCLUSIONS: Our hospital-based programme is capable of positively impacting adolescents' attitudes towards guns and violence. This effect is most pronounced in subjects who already display increased tendencies towards violence. These results suggest that hospitals offer a unique opportunity to address the public health crisis posed by inner-city firearm violence.