CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus (AAV) and 2-Cell Embryo Electroporation.

Genome editing technology is widely used to produce genetically modified animals, including rats. Cytoplasmic or pronuclear injection of DNA repair templates and CRISPR-Cas reagents is the most common delivery method into embryos. However, this type of micromanipulation necessitates access to specialized equipment, is laborious, and requires a certain level of technical skill. Moreover, microinjection techniques often result in lower embryo survival due to the mechanical stress on the embryo. In this protocol, we developed an optimized method to deliver large DNA repair templates to work in conjunction with CRISPR-Cas9 genome editing without the need for microinjection. This protocol combines AAV-mediated DNA delivery of single-stranded DNA donor templates along with the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) by electroporation to modify 2-cell embryos. Using this novel strategy, we have successfully produced targeted knock-in rat models carrying insertion of DNA sequences from 1.2 to 3.0 kb in size with efficiencies between 42% and 90%.

Rising Racial Disparities in Opioid Mortality and Undertreatment of Opioid Use Disorder and Mental Health Comorbidities in Virginia.

Introduction: There were more than 100,000 fatal drug overdoses in the U.S. in 2021 alone. In recent years, there has been a shift in opioid mortality from predominantly White rural communities to Black urban communities. This study aimed to identify the Virginia communities disproportionately affected by the overdose crisis and to better understand the systemic factors contributing to disparities in opioid mortality. Methods: Using the state all-payer claims database, state mortality records, and census data, we created a multivariate model to examine the community-level factors contributing to racial disparities in opioid mortality. We used generalized linear mixed models to examine the associations between socioecologic factors and fatal opioid overdoses, opioid use disorder diagnoses, opioid-related emergency department visits, and mental health diagnoses. Results: Between 2015 and 2020, racial disparities in mortality widened. In 2020, Black males were 1.5 times more likely to die of an opioid overdose than White males (47.3 vs 31.6 per 100,000; p<0.001). The rate of mental health disorders strongly correlated with mortality (ß=0.53, p<0.001). Black individuals are not more likely to be diagnosed with opioid use disorder (ß=0.01, p=0.002) or with mental health disorders (ß= -0.12, p<0.001), despite higher fatal opioid overdoses. Conclusions: There are widening racial disparities in opioid mortality. Untreated mental health disorders are a major risk factor for opioid mortality. Findings show pathways to address inequities, including early linkage to care for mental health and opioid use disorders. This analysis shows the use of comprehensive socioecologic data to identify the precursors to fatal overdoses, which could allow earlier intervention and reallocation of resources in high-risk communities.

Identifying bright spot communities: Socioecological, workforce, and healthcare delivery factors influencing opioid mortality.

Context: There were 50,000 U.S. opioid overdose deaths in 2019. Millions suffer from opioid addiction. Identifying protective factors for low community opioid mortality may have important implications for addressing the opioid epidemic. This study was funded through the Virginia (VA) Department of Medical Assistance Services (DMAS) through a SUPPORT Act Grant. Objective: To identify "Bright Spot" communities in Virginia with protective factors associated with reduced opioid mortality and morbidity. Study Design: Ecologic study. Dataset: Virginia All Payer Claims Database (APCD), Virginia Department of Health (VDH) statewide medical examiner registry, and American Community Survey (ACS). Time Period: 2016-2019; 2019 data cited here. Population Studied: APCD includes VA residents with medical claims through commercial, Medicaid, and Medicare coverage. VDH data includes fatal drug overdoses. ACS surveys all VA residents. Outcome Measures: Primary outcome: fatal opioid overdoses. Secondary outcomes: emergency room visits for overdoses and opioid-related diagnoses, outpatient diagnoses for opioid-related disorder, prescription rate for opioids, and prescription rate for buprenorphine. Results: Opioid mortality was associated with higher rates of community poverty (r=.38, p<.0001) and disability (r=.52, r<.0001). Opioid mortality was associated with inequality, with higher Gini index associated with higher opioid mortality (r=.23, p<.0001). A higher percentage of black residents was associated with increased fatal opioid overdoses (r=.37, p<.0001) and ED visits for overdoses (r=.30, p<.0001). A higher percentage of white residents correlated with increased outpatient visits for opioid use disorder (r=.24, p<.0001) and higher rates of buprenorphine (r=.34, p<.0001) and opioid prescriptions (r=.31, p <.0001). Conclusions: These findings suggest significant racial disparities in opioid outcomes. Communities with a higher percentage of black residents are more likely to have higher opioid mortality and a lower rate of outpatient treatment. This association may be affected by the time period used in the analysis (2015-2019), as nationally there has been an increasing rate of synthetic opioid deaths in Black communities. These measures have been incorporated into a multivariate analysis to identify Bright Spot communities, which will be discussed during the presentation.