Generation of a Commercial-Scale Founder Population of Porcine Reproductive and Respiratory Syndrome Virus Resistant Pigs Using CRISPR-Cas.

Disease resistance genes in livestock provide health benefits to animals and opportunities for farmers to meet the growing demand for affordable, high-quality protein. Previously, researchers used gene editing to modify the porcine CD163 gene and demonstrated resistance to a harmful virus that causes porcine reproductive and respiratory syndrome (PRRS). To maximize potential benefits, this disease resistance trait needs to be present in commercially relevant breeding populations for multiplication and distribution of pigs. Toward this goal, a first-of-its-kind, scaled gene editing program was established to introduce a single modified CD163 allele into four genetically diverse, elite porcine lines. This effort produced healthy pigs that resisted PRRS virus infection as determined by macrophage and animal challenges. This founder population will be used for additional disease and trait testing, multiplication, and commercial distribution upon regulatory approval. Applying CRISPR-Cas to eliminate a viral disease represents a major step toward improving animal health.

Built Environment and SARS-CoV-2 Transmission in Long-Term Care Facilities: Cross-Sectional Survey and Data Linkage.

OBJECTIVES: To describe the built environment in long-term care facilities (LTCF) and its association with introduction and transmission of SARS-CoV-2 infection. DESIGN: Cross-sectional survey with linkage to routine surveillance data. SETTING AND PARTICIPANTS: LTCFs in England caring for adults ≥65 years old, participating in the VIVALDI study (ISRCTN14447421) were eligible. Data were included from residents and staff. METHODS: Cross-sectional survey of the LTCF built environment with linkage to routinely collected asymptomatic and symptomatic SARS-CoV-2 testing and vaccination data between September 1, 2020, and March 31, 2022. We used individual and LTCF level Poisson and Negative Binomial regression models to identify risk factors for 4 outcomes: incidence rate of resident infections and outbreaks, outbreak size, and duration. We considered interactions with variant transmissibility (pre vs post Omicron dominance). RESULTS: A total of 134 of 151 (88.7%) LTCFs participated in the survey, contributing data for 13,010 residents and 17,766 staff. After adjustment and stratification, outbreak incidence (measuring infection introduction) was only associated with SARS-CoV-2 incidence in the community [incidence rate ratio (IRR) for high vs low incidence, 2.84; 95% CI, 1.85-4.36]. Characteristics of the built environment were associated with transmission outcomes and differed by variant transmissibility. For resident infection incidence, factors included number of storeys (0.64; 0.43-0.97) and bedrooms (1.04; 1.02-1.06), and purpose-built vs converted buildings (1.99; 1.08-3.69). Air quality was associated with outbreak size (dry vs just right 1.46; 1.00-2.13). Funding model (0.99; 0.99-1.00), crowding (0.98; 0.96-0.99), and bedroom temperature (1.15; 1.01-1.32) were associated with outbreak duration. CONCLUSIONS AND IMPLICATIONS: We describe previously undocumented diversity in LTCF built environments. LTCFs have limited opportunities to prevent SARS-CoV-2 introduction, which was only driven by community incidence. However, adjusting the built environment, for example by isolating infected residents or improving airflow, may reduce transmission, although data quality was limited by subjectivity. Identifying LTCF built environment modifications that prevent infection transmission should be a research priority.

Allogeneic chimeric antigen receptor-T cells with CRISPR-disrupted programmed death-1 checkpoint exhibit enhanced functional fitness.

BACKGROUND AIMS: Therapeutic disruption of immune checkpoints has significantly advanced the armamentarium of approaches for treating cancer. The prominent role of the programmed death-1 (PD-1)/programmed death ligand-1 axis for downregulating T cell function offers a tractable strategy for enhancing the disease-modifying impact of CAR-T cell therapy. METHODS: To address checkpoint interference, primary human T cells were genome edited with a next-generation CRISPR-based platform (Cas9 chRDNA) by knockout of the PDCD1 gene encoding the PD-1 receptor. Site-specific insertion of a chimeric antigen receptor specific for CD19 into the T cell receptor alpha constant locus was implemented to drive cytotoxic activity. RESULTS: These allogeneic CAR-T cells (CB-010) promoted longer survival of mice in a well-established orthotopic tumor xenograft model of a B cell malignancy compared with identically engineered CAR-T cells without a PDCD1 knockout. The persistence kinetics of CB-010 cells in hematologic tissues versus CAR-T cells without PDCD1 disruption were similar, suggesting the robust initial debulking of established tumor xenografts was due to enhanced functional fitness. By single-cell RNA-Seq analyses, CB-010 cells, when compared with identically engineered CAR-T cells without a PDCD1 knockout, exhibited fewer Treg cells, lower exhaustion phenotypes and reduced dysfunction signatures and had higher activation, glycolytic and oxidative phosphorylation signatures. Further, an enhancement of mitochondrial metabolic fitness was observed, including increased respiratory capacity, a hallmark of less differentiated T cells. CONCLUSIONS: Genomic PD-1 checkpoint disruption in the context of allogeneic CAR-T cell therapy may provide a compelling option for treating B lymphoid malignancies.

Outcomes of SARS-CoV-2 omicron infection in residents of long-term care facilities in England (VIVALDI): a prospective, cohort study.

Background: The SARS-CoV-2 omicron variant (B.1.1.529) is highly transmissible, but disease severity appears to be reduced compared with previous variants such as alpha and delta. We investigated the risk of severe outcomes following infection in residents of long-term care facilities. Methods: We did a prospective cohort study in residents of long-term care facilities in England who were tested regularly for SARS-CoV-2 between Sept 1, 2021, and Feb 1, 2022, and who were participants of the VIVALDI study. Residents were eligible for inclusion if they had a positive PCR or lateral flow device test during the study period, which could be linked to a National Health Service (NHS) number, enabling linkage to hospital admissions and mortality datasets. PCR or lateral flow device test results were linked to national hospital admission and mortality records using the NHS-number-based pseudo-identifier. We compared the risk of hospital admission (within 14 days following a positive SARS-CoV-2 test) or death (within 28 days) in residents who had tested positive for SARS-CoV-2 in the period shortly before omicron emerged (delta-dominant) and in the omicron-dominant period, adjusting for age, sex, primary vaccine course, past infection, and booster vaccination. Variants were confirmed by sequencing or spike-gene status in a subset of samples. Results: 795 233 tests were done in 333 long-term care facilities, of which 159 084 (20·0%) could not be linked to a pseudo-identifier and 138 012 (17·4%) were done in residents. Eight residents had two episodes of infection (>28 days apart) and in these cases the second episode was excluded from the analysis. 2264 residents in 259 long-term care facilities (median age 84·5 years, IQR 77·9-90·0) were diagnosed with SARS-CoV-2, of whom 253 (11·2%) had a previous infection and 1468 (64·8%) had received a booster vaccination. About a third of participants were male. Risk of hospital admissions was markedly lower in the 1864 residents infected in the omicron-period (4·51%, 95% CI 3·65-5·55) than in the 400 residents infected in the pre-omicron period (10·50%, 7·87-13·94), as was risk of death (5·48% [4·52-6·64] vs 10·75% [8·09-14·22]). Adjusted hazard ratios (aHR) also indicated a reduction in hospital admissions (0·64, 95% CI 0·41-1·00; p=0·051) and mortality (aHR 0·68, 0·44-1·04; p=0·076) in the omicron versus the pre-omicron period. Findings were similar in residents with a confirmed variant. Interpretation: Observed reduced severity of the omicron variant compared with previous variants suggests that the wave of omicron infections is unlikely to lead to a major surge in severe disease in long-term care facility populations with high levels of vaccine coverage or natural immunity. Continued surveillance in this vulnerable population is important to protect residents from infection and monitor the public health effect of emerging variants. Funding: UK Department of Health and Social Care.

Severe Acute Respiratory Syndrome Coronavirus 2 Anti-Spike Antibody Levels Following Second Dose of ChAdOx1 nCov-19 or BNT162b2 Vaccine in Residents of Long-term Care Facilities in England (VIVALDI).

General population studies have shown strong humoral response following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination with subsequent waning of anti-spike antibody levels. Vaccine-induced immune responses are often attenuated in frail and older populations, but published data are scarce. We measured SARS-CoV-2 anti-spike antibody levels in long-term care facility residents and staff following a second vaccination dose with Oxford-AstraZeneca or Pfizer-BioNTech. Vaccination elicited robust antibody responses in older residents, suggesting comparable levels of vaccine-induced immunity to that in the general population. Antibody levels are higher after Pfizer-BioNTech vaccination but fall more rapidly compared to Oxford-AstraZeneca recipients and are enhanced by prior infection in both groups.

Robust SARS-CoV-2-specific and heterologous immune responses in vaccine-naïve residents of long-term care facilities who survive natural infection.

We studied humoral and cellular immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 152 long-term care facility staff and 124 residents over a prospective 4-month period shortly after the first wave of infection in England. We show that residents of long-term care facilities developed high and stable levels of antibodies against spike protein and receptor-binding domain. Nucleocapsid-specific responses were also elevated but waned over time. Antibodies showed stable and equivalent levels of functional inhibition against spike-angiotensin-converting enzyme 2 binding in all age groups with comparable activity against viral variants of concern. SARS-CoV-2 seropositive donors showed high levels of antibodies to other beta-coronaviruses but serostatus did not impact humoral immunity to influenza or other respiratory syncytial viruses. SARS-CoV-2-specific cellular responses were similar across all ages but virus-specific populations showed elevated levels of activation in older donors. Thus, survivors of SARS-CoV-2 infection show a robust and stable immunity against the virus that does not negatively impact responses to other seasonal viruses.

Storytelling as a Research Tool Used to Explore Insights and as an Intervention in Public Health: A Systematic Narrative Review.

Objectives: Studies of storytelling (ST) used as a research tool to extract information and/or as an intervention to effect change in the public knowledge, attitudes, and behavior/practice (KAB/P) were sought and analyzed. Methods: Medline, EMBASE, PsycINFO, ERIC, Web of Science, Art and Humanities database, Scopus, and Google Scholar were searched, and a basic and broad quantitative analysis was performed, followed by an in-depth narrative synthesis of studies on carefully selected topics. Results: From this search, 3,077 studies were identified. 145 studies entered quantitative analysis [cancer and cancer screening (32/145), HIV (32/145), mental health (10/145), vaccination (8/145), and climate change (3/145)]. Ten studies entered final analysis [HIV/AIDs (5), climate change (1), sexual health (3), and croup (1)]. ST techniques included digital ST (DST), written ST, verbal ST, and use of professional writers. Of the ten studies, seven used ST to change KAB/P; the remainder used ST to extract insights. Follow-up and evaluation were very limited. Conclusion: ST reveals insights and serves as an intervention in public health. Benefits of ST largely outweigh the limitations, but more follow-up/evaluation is needed. ST should play a more significant role in tackling public health issues. PROSPERO registration number: CRD42019124704.

Profile of humoral and cellular immune responses to single doses of BNT162b2 or ChAdOx1 nCoV-19 vaccines in residents and staff within residential care homes (VIVALDI): an observational study.

BACKGROUND: Residents of long-term care facilities (LTCFs) have been prioritised for COVID-19 vaccination because of the high COVID-19 mortality in this population. Several countries have implemented an extended interval of up to 12 weeks between the first and second vaccine doses to increase population coverage of single-dose vaccination. We aimed to assess the magnitude and quality of adaptive immune responses following a single dose of COVID-19 vaccine in LTCF residents and staff. METHODS: From the LTCFs participating in the ongoing VIVALDI study (ISRCTN14447421), staff and residents who had received a first dose of COVID-19 vaccine (BNT162b2 [tozinameran] or ChAdOx1 nCoV-19), had pre-vaccination and post-vaccination blood samples (collected between Dec 11, 2020, and Feb 16, 2021), and could be linked to a pseudoidentifier in the COVID-19 Data Store were included in our cohort. Past infection with SARS-CoV-2 was defined on the basis of nucleocapsid-specific IgG antibodies being detected through a semiquantitative immunoassay, and participants who tested positive on this assay after but not before vaccination were excluded from the study. Processed blood samples were assessed for spike-specific immune responses, including spike-specific IgG antibody titres, T-cell responses to spike protein peptide mixes, and inhibition of ACE2 binding by spike protein from four variants of SARS-CoV-2 (the original strain as well as the B.1.1.7, B.1.351, and P.1 variants). Responses before and after vaccination were compared on the basis of age, previous infection status, role (staff or resident), and time since vaccination. FINDINGS: Our cohort comprised 124 participants from 14 LTCFs: 89 (72%) staff (median age 48 years [IQR 35·5-56]) and 35 (28%) residents (87 years [77-90]). Blood samples were collected a median 40 days (IQR 25-47; range 6-52) after vaccination. 30 (24%) participants (18 [20%] staff and 12 [34%] residents) had serological evidence of previous SARS-CoV-2 infection. All participants with previous infection had high antibody titres following vaccination that were independent of age (r s=0·076, p=0·70). In participants without evidence of previous infection, titres were negatively correlated with age (r s=-0·434, p<0·0001) and were 8·2-times lower in residents than in staff. This effect appeared to result from a kinetic delay antibody generation in older infection-naive participants, with the negative age correlation disappearing only in samples taken more than 42 days post-vaccination (r s=-0·207, p=0·20; n=40), in contrast to samples taken after 0-21 days (r s=-0·774, p=0·0043; n=12) or 22-42 days (r s=-0·437, p=0·0034; n=43). Spike-specific cellular responses were similar between older and younger participants. In infection-naive participants, antibody inhibition of ACE2 binding by spike protein from the original SARS-CoV-2 strain was negatively correlated with age (r s=-0·439, p<0·0001), and was significantly lower against spike protein from the B.1.351 variant (median inhibition 31% [14-100], p=0·010) and the P.1 variant (23% [14-97], p<0·0001) than against the original strain (58% [27-100]). By contrast, a single dose of vaccine resulted in around 100% inhibition of the spike-ACE2 interaction against all variants in people with a history of infection. INTERPRETATION: History of SARS-CoV-2 infection impacts the magnitude and quality of antibody response after a single dose of COVID-19 vaccine in LTCF residents. Residents who are infection-naive have delayed antibody responses to the first dose of vaccine and should be considered for an early second dose where possible. FUNDING: UK Government Department of Health and Social Care.

Feasibility study of hospital antimicrobial stewardship analytics using electronic health records.

BACKGROUND: Hospital antimicrobial stewardship (AMS) programmes are multidisciplinary initiatives to optimize antimicrobial use. Most hospitals depend on time-consuming manual audits to monitor clinicians' prescribing. But much of the information needed could be sourced from electronic health records (EHRs). OBJECTIVES: To develop an informatics methodology to analyse characteristics of hospital AMS practice using routine electronic prescribing and laboratory records. METHODS: Feasibility study using electronic prescribing, laboratory and clinical coding records from adult patients admitted to six specialities at Queen Elizabeth Hospital, Birmingham, UK (September 2017-August 2018). The study involved: (i) a review of AMS standards of care; (ii) their translation into concepts measurable from commonly available EHRs; and (iii) a pilot application in an EHR cohort study (n = 61679 admissions). RESULTS: We developed data modelling methods to characterize antimicrobial use (antimicrobial therapy episode linkage methods, therapy table, therapy changes). Prescriptions were linked into antimicrobial therapy episodes (mean 2.4 prescriptions/episode; mean length of therapy 5.8 days), enabling several actionable findings. For example, 22% of therapy episodes for low-severity community-acquired pneumonia were congruent with prescribing guidelines, with a tendency to use broader-spectrum antibiotics. Analysis of therapy changes revealed IV to oral therapy switching was delayed by an average 3.6 days (95% CI: 3.4-3.7). Microbial cultures were performed prior to treatment initiation in just 22% of antibacterial prescriptions. The proposed methods enabled fine-grained monitoring of AMS practice down to specialities, wards and individual clinical teams by case mix, enabling more meaningful peer comparison. CONCLUSIONS: It is feasible to use hospital EHRs to construct rapid, meaningful measures of prescribing quality with potential to support quality improvement interventions (audit/feedback to prescribers), engagement with front-line clinicians on optimizing prescribing, and AMS impact evaluation studies.

Incidence of SARS-CoV-2 infection according to baseline antibody status in staff and residents of 100 long-term care facilities (VIVALDI): a prospective cohort study.

BACKGROUND: SARS-CoV-2 infection represents a major challenge for long-term care facilities (LTCFs) and many residents and staff are seropositive following persistent outbreaks. We aimed to investigate the association between the SARS-CoV-2 antibody status at baseline and subsequent infection in this population. METHODS: We did a prospective cohort study of SARS-CoV-2 infection in staff (aged <65 years) and residents (aged >65 years) at 100 LTCFs in England between Oct 1, 2020, and Feb 1, 2021. Blood samples were collected between June and November, 2020, at baseline, and 2 and 4 months thereafter and tested for IgG antibodies to SARS-CoV-2 nucleocapsid and spike proteins. PCR testing for SARS-CoV-2 was done weekly in staff and monthly in residents. Cox regression was used to estimate hazard ratios (HRs) of a PCR-positive test by baseline antibody status, adjusted for age and sex, and stratified by LTCF. FINDINGS: 682 residents from 86 LCTFs and 1429 staff members from 97 LTCFs met study inclusion criteria. At baseline, IgG antibodies to nucleocapsid were detected in 226 (33%) of 682 residents and 408 (29%) of 1429 staff members. 93 (20%) of 456 residents who were antibody-negative at baseline had a PCR-positive test (infection rate 0·054 per month at risk) compared with four (2%) of 226 residents who were antibody-positive at baseline (0·007 per month at risk). 111 (11%) of 1021 staff members who were antibody-negative at baseline had PCR-positive tests (0·042 per month at risk) compared with ten (2%) of 408 staff members who were antibody-positive staff at baseline (0·009 per month at risk). The risk of PCR-positive infection was higher for residents who were antibody-negative at baseline than residents who were antibody-positive at baseline (adjusted HR [aHR] 0·15, 95% CI 0·05-0·44, p=0·0006), and the risk of a PCR-positive infection was also higher for staff who were antibody-negative at baseline compared with staff who were antibody-positive at baseline (aHR 0·39, 0·19-0·82; p=0·012). 12 of 14 reinfected participants had available data on symptoms, and 11 of these participants were symptomatic. Antibody titres to spike and nucleocapsid proteins were comparable in PCR-positive and PCR-negative cases. INTERPRETATION: The presence of IgG antibodies to nucleocapsid protein was associated with substantially reduced risk of reinfection in staff and residents for up to 10 months after primary infection. FUNDING: UK Government Department of Health and Social Care.

Landscape Simplification Modifies Trap-Nesting Bee and Wasp Communities in the Subtropics.

(1) Background: Landscape simplification is a major threat to bee and wasp conservation in the tropics, but reliable, long-term population data are lacking. We investigated how community composition, diversity, and abundance of tropical solitary bees and wasps change with landscape simplification (plant diversity, plant richness, distance from forest, forest cover, and land use type) and season. (2) Methods: We installed 336 timber and cob trap nests in four complex forests and three simplified orchards within the subtropical biodiversity hotspot of south-east Queensland, Australia. Trap nests were replaced every season for 23 months and all emergents identified. (3) Results: We identified 28 wasp species and 13 bee species from 2251 brood cells. Bee and wasp community composition changed with landscape simplification such that large, ground-nesting, and spider-hunting species were present in all landscapes, while those with specialist resource requirements and (clepto) parasitoids were present only in complex landscapes. Abundance and diversity of bees and wasps were unaffected by landscape simplification but increased with rainfall. (4) Conclusions: This study highlights the need for multi-year studies incorporating nuanced measures such as composition with a focus on functional diversity to detect changes bee and wasp populations.

Interventions to improve appropriate antibiotic prescribing in long-term care facilities: a systematic review.

BACKGROUND: Overuse of antibiotics has contributed to antimicrobial resistance; a growing public health threat. In long-term care facilities, levels of inappropriate prescribing are as high as 75%. Numerous interventions targeting long-term care facilities' antimicrobial stewardship have been reported with varying, and largely unexplained, effects. Therefore, this review aimed to apply behavioural science frameworks to specify the component behaviour change techniques of stewardship interventions in long-term care facilities and identify those components associated with improved outcomes. METHOD: A systematic review (CRD42018103803) was conducted through electronic database searches. Two behavioural science frameworks, the Behaviour Change Wheel and Behaviour Change Technique Taxonomy were used to classify intervention descriptions into intervention types and component behaviour change techniques used. Study design and outcome heterogeneity prevented meta-analysis and meta-regression. Interventions were categorised as 'very promising' (all outcomes statistically significant), 'quite promising' (some outcomes statistically significant), or 'not promising' (no outcomes statistically significant). 'Promise ratios' (PR) were calculated for identified intervention types and behaviour change techniques by dividing the number of (very or quite) promising interventions featuring the intervention type or behaviour change technique by the number of interventions featuring the intervention type or behaviour change technique that were not promising. Promising intervention types and behaviour change techniques were defined as those with a PR ≥ 2. RESULTS: Twenty studies (of19 interventions) were included. Seven interventions (37%) were 'very promising', eight 'quite promising' (42%) and four 'not promising' (21%). Most promising intervention types were 'persuasion' (n = 12; promise ratio (PR) = 5.0), 'enablement' (n = 16; PR = 4.33) and 'education' (n = 19; PR = 3.75). Most promising behaviour change techniques were 'feedback on behaviour' (n = 9; PR = 8.0) and 'restructuring the social environment' (e.g. staff role changes; n = 8; PR = 7.0). CONCLUSION: Systematic identification of the active ingredients of antimicrobial stewardship in long-term care facilities was facilitated through the application of behavioural science frameworks. Incorporating environmental restructuring and performance feedback may be promising intervention strategies for antimicrobial stewardship interventions within long-term care facilities.

Harnessing type I CRISPR-Cas systems for genome engineering in human cells.

Type I CRISPR-Cas systems are the most abundant adaptive immune systems in bacteria and archaea1,2. Target interference relies on a multi-subunit, RNA-guided complex called Cascade3,4, which recruits a trans-acting helicase-nuclease, Cas3, for target degradation5-7. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain8-11 and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50%. FokI-Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade-Cas3 complex in human cells resulted in targeted deletions of up to ~200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR-Cas systems can be harnessed for genome engineering applications in eukaryotic cells.

Interspecific homeostatic regulation and growth across aquatic invertebrate detritivores: a test of ecological stoichiometry theory.

Across resource quality gradients, primary consumers must regulate homeostasis and release of nutrients to optimize growth and fitness. Based primarily on internal body composition, the ecological stoichiometry theory (EST) offers a framework to generalize interspecific patterns of these responses, yet the predictions and underlying assumptions of EST remain poorly tested across many species. We used controlled laboratory feeding experiments to measure homeostasis, nutrient release, and growth across seven field-collected aquatic invertebrate detritivore taxa fed wide resource carbon:nitrogen (C:N) and carbon:phosphorus (C:P) gradients. We found that most invertebrates exhibited strict stoichiometric homeostasis (average 1/H = - 0.018 and 0.026 for C:N and C:P, respectively), supporting assumptions of EST. However, the stoichiometry of new tissue production during growth intervals (growth stoichiometry) deviated - 30 to + 54% and - 145 to + 74% from initial body C:N and C:P, respectively, and across species, growth stoichiometry was not correlated with initial body stoichiometry. Notably, smaller non- and hemimetabolous invertebrates exhibited low, decreasing growth C:N and C:P, whereas larger holometabolous invertebrates exhibited high, often increasing growth C:N and C:P. Despite predictions of EST, interspecific sensitivity of egestion stoichiometry and growth rates to the resource gradient were weakly related to internal body composition across species. While the sensitivity of these patterns differed across taxa, such differences carried a weak phylogenetic signal and were not well predicted by EST. Our findings suggest that traits beyond internal body composition, such as feeding behavior, selective assimilation, and ontogeny, are needed to generalize interspecific patterns in consumer growth and nutrient release across resource quality gradients.

NmeCas9 is an intrinsically high-fidelity genome-editing platform.

BACKGROUND: The development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wildtype SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs are considerably smaller and therefore better suited for viral delivery. RESULTS: Here we show that wildtype NmeCas9, when programmed with guide sequences of the natural length of 24 nucleotides, exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5'-N4GATT-3'), for NmeCas9 genome editing in human cells. CONCLUSIONS: Our results show that NmeCas9 is a naturally high-fidelity genome-editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering.

Mapping the genomic landscape of CRISPR-Cas9 cleavage.

RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.

Cost-effectiveness of national mandatory screening of all admissions to English National Health Service hospitals for meticillin-resistant Staphylococcus aureus: a mathematical modelling study.

BACKGROUND: In December, 2010, National Health Service (NHS) England introduced national mandatory screening of all admissions for meticillin-resistant Staphylococcus aureus (MRSA). We aimed to assess the effectiveness and cost-effectiveness of this policy, from a regional or national health-care decision makers' perspective, compared with alternative screening strategies. METHODS: We used an individual-based dynamic transmission model parameterised with national MRSA audit data to assess the effectiveness and cost-effectiveness of admission screening of patients in English NHS hospitals compared with five alternative strategies (including no screening, checklist-activated screening, and high-risk specialty-based screening), accompanied by patient isolation and decolonisation, over a 5 year time horizon. We evaluated strategies for different NHS hospital types (acute, teaching, and specialist), MRSA prevalence, and transmission potentials using probabilistic sensitivity analyses. FINDINGS: Compared with no screening, mean cost per quality-adjusted life-year (QALY) of screening all admissions was £89,000-148,000 (range £68,000-222,000), and this strategy was consistently more costly and less effective than alternatives for all hospital types. At a £30,000/QALY willingness-to-pay threshold and current prevalence, only the no-screening strategy was cost effective. The next best strategies were, in acute and teaching hospitals, targeting of high-risk specialty admissions (30-40% chance of cost-effectiveness; mean incremental cost-effectiveness ratios [ICERs] £45,200 [range £35,300-61,400] and £48,000/QALY [£34,600-74,800], respectively) and, in specialist hospitals, screening these patients plus risk-factor-based screening of low-risk specialties (a roughly 20% chance of cost-effectiveness; mean ICER £62,600/QALY [£48,000-89,400]). As prevalence and transmission increased, targeting of high-risk specialties became the optimum strategy at the NHS willingness-to-pay threshold (£30,000/QALY). Switching from screening all admissions to only high-risk specialty admissions resulted in a mean reduction in total costs per year (not considering uncertainty) of £2·7 million per acute hospital, £2·9 million per teaching, and £474,000 per specialist hospital for a minimum rise in infections (about one infection per year per hospital). INTERPRETATION: Our results show that screening all admissions for MRSA is unlikely to be cost effective in England at the current NHS willingness-to-pay threshold, and our findings informed modified guidance to NHS England in 2014. Screening admissions to high-risk specialties is likely to represent better resource use in terms of cost per QALY gained. FUNDING: UK Department of Health.

Impact of a cis-associated gene expression SNP on chromosome 20q11.22 on bipolar disorder susceptibility, hippocampal structure and cognitive performance.

BACKGROUND: Bipolar disorder is a highly heritable polygenic disorder. Recent enrichment analyses suggest that there may be true risk variants for bipolar disorder in the expression quantitative trait loci (eQTL) in the brain. AIMS: We sought to assess the impact of eQTL variants on bipolar disorder risk by combining data from both bipolar disorder genome-wide association studies (GWAS) and brain eQTL. METHOD: To detect single nucleotide polymorphisms (SNPs) that influence expression levels of genes associated with bipolar disorder, we jointly analysed data from a bipolar disorder GWAS (7481 cases and 9250 controls) and a genome-wide brain (cortical) eQTL (193 healthy controls) using a Bayesian statistical method, with independent follow-up replications. The identified risk SNP was then further tested for association with hippocampal volume (n = 5775) and cognitive performance (n = 342) among healthy individuals. RESULTS: Integrative analysis revealed a significant association between a brain eQTL rs6088662 on chromosome 20q11.22 and bipolar disorder (log Bayes factor = 5.48; bipolar disorder P = 5.85 × 10(-5)). Follow-up studies across multiple independent samples confirmed the association of the risk SNP (rs6088662) with gene expression and bipolar disorder susceptibility (P = 3.54 × 10(-8)). Further exploratory analysis revealed that rs6088662 is also associated with hippocampal volume and cognitive performance in healthy individuals. CONCLUSIONS: Our findings suggest that 20q11.22 is likely a risk region for bipolar disorder; they also highlight the informative value of integrating functional annotation of genetic variants for gene expression in advancing our understanding of the biological basis underlying complex disorders, such as bipolar disorder.

Growth and stoichiometry of a common aquatic detritivore respond to changes in resource stoichiometry.

Consumer growth determines the quantity of nutrients transferred through food webs. The extent to which leaf composition and consumer physiology interact to constrain consumer production is not well understood. For example, detritivore growth, and thus material transfer, could change with detrital elemental composition. Detrital type and associated microbial biofilms can mediate the amount and rate of detritus consumed and used towards growth. Detritivore body stoichiometry or the threshold elemental ratio, the food ratio resulting in optimal growth, may predict taxon-specific growth response to stoichiometrically-altered detritus. Empirical measures of detritivore growth responses across a range of detrital stoichiometry are rare. We fed a common detritivore, Tipula abdominalis, maple or oak leaves that spanned a gradient of carbon:phosphorus (C:P) to examine how leaf identity and C:P interact to influence growth, consumption, assimilation efficiencies, and post-assimilatory processes. Tipula abdominalis growth and consumption varied with leaf type and stoichiometry. Individuals fed oak grew faster and ate more compared to individuals fed maple. Individuals fed maple grew faster and ate more as leaf C:P decreased. All individuals lost most of the material they assimilated through respiration and excretion regardless of leaf type or leaf stoichiometry. Consumption and growth rates of T. abdominalis increased with maple nutrient enrichment, but not oak, indicating leaf-specific nutrient enrichment affected leaf palatability. Slightly non-homeostatic T. abdominalis C:P was maintained by varied consumption, carbon assimilation, and P excretion. Our study underlines the importance of how detritivore consumption and post-assimilatory processing could influence whole-stream material storage and nutrient cycling in detrital-based ecosystems.