Efficient Homology-Directed Repair with Circular Single-Stranded DNA Donors.

While genome editing has been revolutionized by the advent of CRISPR-based nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair (HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer from low HDR efficiencies in many cell types, as well as integration at unintended sites. In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with minimal off-target integration.

Allelic variation in class I HLA determines CD8 T cell repertoire shape and cross-reactive memory responses to SARS-CoV-2.

Effective presentation of antigens by human leukocyte antigen (HLA) class I molecules to CD8 T cells is required for viral elimination and generation of long-term immunological memory. In this study, we applied a single-cell, multiomic technology to generate a unified ex vivo characterization of the CD8 T cell response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) across four major HLA class I alleles. We found that HLA genotype conditions key features of epitope specificity, TCRα/β sequence diversity, and the utilization of pre-existing SARS-CoV-2-reactive memory T cell pools.

Valves Are a Conserved Feature of the Zebrafish Lymphatic System.

The lymphatic system comprises blind-ended tubes that collect interstitial fluid and return it to the circulatory system. In mammals, unidirectional lymphatic flow is driven by muscle contraction working in conjunction with valves. Accordingly, defective lymphatic valve morphogenesis results in backflow leading to edema.

Precise therapeutic gene correction by a simple nuclease-induced double-stranded break.

Current programmable nuclease-based methods (for example, CRISPR-Cas9) for the precise correction of a disease-causing genetic mutation harness the homology-directed repair pathway. However, this repair process requires the co-delivery of an exogenous DNA donor to recode the sequence and can be inefficient in many cell types. Here we show that disease-causing frameshift mutations that result from microduplications can be efficiently reverted to the wild-type sequence simply by generating a DNA double-stranded break near the centre of the duplication.

Highly efficient therapeutic gene editing of human hematopoietic stem cells.

Re-expression of the paralogous γ-globin genes (HBG1/2) could be a universal strategy to ameliorate the severe β-globin disorders sickle cell disease (SCD) and β-thalassemia by induction of fetal hemoglobin (HbF, αγ). Previously, we and others have shown that core sequences at the BCL11A erythroid enhancer are required for repression of HbF in adult-stage erythroid cells but are dispensable in non-erythroid cells. CRISPR-Cas9-mediated gene modification has demonstrated variable efficiency, specificity, and persistence in hematopoietic stem cells (HSCs).

Enhanced Cas12a editing in mammalian cells and zebrafish.

Type V CRISPR-Cas12a systems provide an alternate nuclease platform to Cas9, with potential advantages for specific genome editing applications. Here we describe improvements to the Cas12a system that facilitate efficient targeted mutagenesis in mammalian cells and zebrafish embryos. We show that engineered variants of Cas12a with two different nuclear localization sequences (NLS) on the C terminus provide increased editing efficiency in mammalian cells.

Cryptosporidiosis Outbreak Investigation in a Canadian Correctional Facility Using Novel Case Finding Tools.

Eliminating Cryptosporidium oocysts is particularly challenging in prison environments, and limited information is available on best practices for outbreak control. This report aims to expand the evidence base through the lessons learned from an outbreak at a Canadian federal penitentiary in 2013. The outbreak investigation methods included inmate and staff case finding, hypothesis-generating questionnaires, and environmental investigation.

Determination of ubiquitin fitness landscapes under different chemical stresses in a classroom setting.

Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations.

Estimating the global abundance of ground level presence of particulate matter (PM2.5).

With the increasing awareness of the health impacts of particulate matter, there is a growing need to comprehend the spatial and temporal variations of the global abundance of ground level airborne particulate matter with a diameter of 2.5 microns or less (PM2.5).

Viewing protein fitness landscapes through a next-gen lens.

High-throughput sequencing has enabled many powerful approaches in biological research. Here, we review sequencing approaches to measure frequency changes within engineered mutational libraries subject to selection. These analyses can provide direct estimates of biochemical and fitness effects for all individual mutations across entire genes (and likely compact genomes in the near future) in genetically tractable systems such as microbes, viruses, and mammalian cells.

Interferon-gamma release assays piloted as a latent tuberculous infection screening tool in Canadian federal inmates.

Background: Interferon-gamma release assays (IGRAs) may be useful in diagnosing latent tuberculous infection (LTBI) in inmates; however, published experience in these settings is limited.

Objective: To identify variables associated with IGRA positivity among Canadian federal inmates with positive tuberculin skin test (TST) results.

Design: On intake, TST-positive (≥10 mm) inmates were offered an IGRA (QuantiFERON(®)-TB Gold), and demographic and historical data were collected.

Systematic exploration of ubiquitin sequence, E1 activation efficiency, and experimental fitness in yeast.

The complexity of biological interaction networks poses a challenge to understanding the function of individual connections in the overall network. To address this challenge, we developed a high-throughput reverse engineering strategy to analyze how thousands of specific perturbations (encompassing all point mutations in a central gene) impact both a specific edge (interaction to a directly connected node) and an overall network function. We analyzed the effects of ubiquitin mutations on activation by the E1 enzyme and compared these to effects on yeast growth rate.

Resistance to vemurafenib resulting from a novel mutation in the BRAFV600E kinase domain.

Resistance to the BRAF inhibitor vemurafenib poses a significant problem for the treatment of BRAFV600E-positive melanomas. It is therefore critical to prospectively identify all vemurafenib resistance mechanisms prior to their emergence in the clinic. The vemurafenib resistance mechanisms described to date do not result from secondary mutations within BRAFV600E.

Analyses of the effects of all ubiquitin point mutants on yeast growth rate.

The amino acid sequence of a protein governs its function. We used bulk competition and focused deep sequencing to investigate the effects of all ubiquitin point mutants on yeast growth rate. Many aspects of ubiquitin function have been carefully studied, which enabled interpretation of our growth analyses in light of a rich structural, biophysical and biochemical knowledge base.

Fitness analyses of all possible point mutations for regions of genes in yeast.

Deep sequencing can accurately measure the relative abundance of hundreds of mutations in a single bulk competition experiment, which can give a direct readout of the fitness of each mutant. Here we describe a protocol that we previously developed and optimized to measure the fitness effects of all possible individual codon substitutions for 10-aa regions of essential genes in yeast. Starting with a conditional strain (i.

Treatment of bipolar disorders and metabolic syndrome: implications for primary care.

Recognition of the prevalence of mood disorders and increased availability of medication options have led to calls for treating bipolar disorders in the primary care setting. Second-generation antipsychotic medications (SGAs) were initially lauded for treating bipolar disorders because of their efficacy and perceived safety relative to first-generation antipsychotic medications. Metabolic syndrome is a constellation of risk factors for cardiovascular disease and type 2 diabetes mellitus, which may emerge when treating bipolar disorders with SGAs.

Sexual harassment: early adolescents' self-reports of experiences and acceptance.

Considerable attention has been focused on sexual harassment experiences and attitudes of older adolescents and adults. Recently, educational and judicial institutions have recognized that harassment also occurs among junior and senior high school students. The primary aim of this project was to gather information regarding early adolescents' experiences with and acceptance of sexual harassment behaviors.

Imaginary audience behavior in older adolescent females.

The purpose of this study was to explore the extent of imaginary audience behavior in older adolescent females to ascertain if such behavior continues beyond the years of early and middle adolescence. Subjects were 112 freshman females enrolled in a midwestern university during their first semester. Results indicated that college freshman displayed a greater degree of imaginary audience behavior than did younger adolescents when compared to scores reported by Elkind and Bowen (1979).

Sibling violence and agonistic interactions among middle adolescents.

Two hundred seventy-two high school juniors and seniors were surveyed regarding their experiences as victims and perpetrators of sibling violence and agonistic interactions with their closest-spaced siblings. Findings support earlier reports that siblings engage in a variety of violent acts directed toward one another and that there are few differences between males and females as to the extent of the behaviors. Subjects reported experiences of negative physical and verbal interactions less frequently than did younger subjects in previous studies, suggesting the need to further examine age-related differences in sibling violence.

AIDS: late adolescents' knowledge and its influence on sexual behavior.

Approximately 300 late adolescents were surveyed to assess their knowledge of AIDS, and to determine if and how their sexual behavior had changed as a result. Findings suggested that late adolescents were quite knowledgeable regarding AIDS and its transmission; however, only about one-third had altered their sexual behavior as a result of fear of the disease. Findings are discussed in terms of the need for AIDS education, and recommendations are presented.

Defining child maltreatment: ratings of parental behaviors.

This study investigated adolescents' assessment of various forms of child abuse and neglect. Participants (N = 204) rated the seriousness of vignettes describing a variety of parental actions relative to the degree of harm each was perceived to have on a child's welfare. Adolescents' responses were compared to those of community members and professionals as reported by Giovannoni and Becerra (1979).

Age-appropriate behaviors: a comparison of three generations of females.

The purpose of this study was to examine differences in older adolescent and adult females' perceptions of age-appropriate behavior in adults. Subjects were three generations of maternally related females (95 older adolescents, 78 mothers, 83 grandmothers) who were administered a questionnaire designed to assess age-appropriate behaviors in three broad categories: recreation, occupation/career, and family. Major findings were that older adolescents were most tolerant of behaviors performed by individuals at varying ages while older women most closely ascribed to age constraints, and that the greatest agreement on appropriateness of age constraints concerned family relationships while least agreement concerned recreational activities.

Loneliness among late adolescents.

The extent of loneliness among late adolescents was assessed by means of the Revised UCLA Loneliness Scale. Findings suggest that although moderate loneliness was reported by most participants, a number of young people evidenced a high degree of loneliness. A comparison of lonely and nonlonely adolescents yielded relatively few significant differences.

Conventional warfare and United States military involvement in Latin America: early adolescents' views.

Early adolescents' views concerning conventional war were assessed in order to better understand the thinking of today's youth, and to determine similarities and differences of the views expressed by adolescents 15 years ago. Three hundred sixty-two seventh-grade students were surveyed regarding conventional warfare in general and United States military involvement in Latin America specifically. Results suggest participants were somewhat more accepting of war in 1986, and that many were skeptical of President Reagan's honesty and wisdom on the issue of United States military involvement in Latin America.