Phosphonoacetate Modifications Enhance the Stability and Editing Yields of Guide RNAs for Cas9 Editors.

CRISPR gene editing and control systems continue to emerge and inspire novel research and clinical applications. Advances in CRISPR performance such as optimizing the duration of activity in cells, tissues, and organisms, as well as limiting off-target activities, have been extremely important for expanding the utility of CRISPR-based systems. By investigating the effects of various chemical modifications in guide RNAs (gRNAs) at defined positions and combinations, we find that 2'-O-methyl-3'-phosphonoacetate (MP) modifications can be substantially more effective than 2'-O-methyl-3'-phosphorothioate (MS) modifications at the 3' ends of single-guide RNAs (sgRNAs) to promote high editing yields, in some instances showing an order of magnitude higher editing yield in human cells. MP-modified 3' ends are especially effective at promoting the activity of guide RNAs cotransfected with Cas messenger RNA (mRNA), as the gRNA must persist in cells until the Cas protein is expressed. We demonstrate such an MP enhancement for sgRNAs cotransfected with a BE4 mRNA for cytidine base editing and also demonstrate that MP at the 3' ends of prime editing guide RNAs (pegRNAs) cotransfected with PE2 mRNA can promote maximal prime editing yields. In the presence of serum, sgRNAs with MP-modified 3' ends showed marked improvements in editing efficiency over sgRNAs with MS-modified 3' ends codelivered with Cas9 mRNA and showed more modest improvements at enhancing the activity of transfected ribonucleoprotein (RNP) complexes. Our results suggest that MP should be considered as a performance-enhancing modification for the 3' ends of synthetic gRNAs, especially in situations where the guide RNAs may be susceptible to exonuclease-mediated degradation.

High throughput screen for the improvement of inducible promoters for tumor microenvironment cues.

Cancer immunotherapies are highly potent and are gaining wide clinical usage. However, severe side effects require focusing effector immune cell activities on the tumor microenvironment (TME). We recently developed a chimeric antigen receptor tumor-induced vector (CARTIV), a synthetic promoter activated by TME factors. To improve CARTIV functions including background, activation levels, and synergism, we screened a library of promoters with variations in key positions. Here, we present a screening method involving turning ON/OFF stimulating TNFα and IFNγ cytokines, followed by sequential cell sorting. Sequencing of enriched promoters identified seventeen candidates, which were cloned and whose activities were then validated, leading to the identification of two CARTIVs with lower background and higher induction. We further combined a third hypoxia element with the two-factor CARTIV, demonstrating additional modular improvement. Our study presents a method of fine-tuning synthetic promoters for desired immunotherapy needs.

Effects of Workplace-Related Factors on the Prevalence of Fibromyalgia among Israeli Kindergarten Teachers.

Background: Fibromyalgia syndrome (FMS), a chronic widespread pain disorder, has been associated with various models of stress, including those that are workplace-related. In a previous study, we have documented the significantly increased prevalence of FMS among schoolteachers, as well as correlating symptoms with stressful workplace-related factors. In the current study, we have focused on the specific population of kindergarten teachers and attempted to document both the prevalence of FMS symptoms among this group and the association with stress and symptoms of posttrauma. Methods: All participants in the study were working as kindergarten teachers in Israel at the time of the study. Participants responded to a questionnaire documenting FMS symptom, which included the widespread pain index (WPI) and symptom severity scale (SSS), which together constitute the suggested American College of Rheumatology (ACR) FMS diagnostic criteria. Additional items on the questionnaire documented work motivation and performance, the occurrence of workplace-related stressful events, and the presence of posttraumatic symptoms. Results: 242 participants were recruited to the current study, including 239 (98.8%) females and 3 (1.2%) males. 62 individuals (25.6%) were found to fulfill ACR FMS criteria. Significant differences in work performance were found between teachers fulfilling FMS criteria compared with those not fulfilling criteria. Thus, FMS-positive teachers reported significantly higher rates of missing workdays, leaving work early, and a lower quality of interaction with children in the kindergarten and with peers and supervisors. Motivation to work was also significantly lower among these individuals. The widespread pain index (WPI) and symptom severity scale (SSS), which together constitute the components of the FMS diagnostic criteria, were positively correlated with both stress and posttraumatic symptoms. In addition, widespread pain, disordered sleep, difficulty with concentration, and other FMS symptoms were strongly correlated with many specific stressful factors at the workplace, including the number of children in the kindergarten, interaction with parents, lack of optimal physical conditions in the classrooms, and various demands on behalf of the educational system. Conclusion: FMS symptoms were found to be highly prevalent among Israeli kindergarten teachers, at a rate that greatly exceeds the prevalence in the general Israeli population. Stressful work-related events appear to be positively associated with the occurrence of FMS symptoms and may serve as triggers for their development. Healthcare professionals treating individuals engaged in this occupation should be vigilant for the occurrence of symptoms that are clinically associated with FMS and overlapping functional disorders.

Evacuation of a mental health center during a forest fire in Israel.

Tirat Carmel Mental Health Center was successfully evacuated in December 2010 during a ravaging forest fire in the nearby Carmel Mountains. A total of 228 patients were successfully evacuated from the center within 45 minutes. No fatalities or injuries associated with the evacuation occurred. We believe that the efficient functioning of the administrative and medical staff provides a replicable model that can contribute to the level of awareness and readiness of hospital staff members for natural and manmade disasters.