A Facile, Transfection-Free Approach to siRNA Delivery in In Vitro 3D Spheroid Models.

Cell culture has long been essential for preclinical modeling of human development and disease. However, conventional two-dimensional (2D) cell culture fails to faithfully model the complexity found in vivo, and novel drug candidates that show promising results in 2D models often do not translate to the clinic. More recently, three-dimensional (3D) cell culture models have gained popularity owing to their greater physiological relevance to in vivo biology. In particular, 3D spheroid models are becoming widely used due to their ability to mimic solid tumors, both in architecture and gradation of nutrients distributed from the outer, proliferative layers into the inner, quiescent layers of cells. Similar to in vivo tumors, cell lines grown in 3D spheroid models tend to be more resistant to antitumor drug treatments than their 2D cultured counterparts, though distinct signaling pathways and gene targets conferring this resistance have yet to be fully explored. RNA interference (RNAi) is an effective tool to elucidate gene function and discover novel druggable targets in 2D models; however, only a few studies have successfully performed RNAi in complex 3D models to date. Here, we demonstrate efficient RNAi-mediated knockdown using "transfection-free" Dharmacon Accell siRNAs in three spheroid culture models, in the presence or absence of the extracellular matrix. This methodology has the potential to be scaled up for complex arrayed screening experiments, which may aid in the identification of novel druggable targets with greater clinical relevance than those identified in 2D experiments. © 2024 Dharmacon, Inc. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of 3D spheroids in matrix-free ULA plates Alternate Protocol 1: Generation of Matrigel matrix-embedded 3D spheroids Alternate Protocol 2: Generation of GrowDex hydrogel-embedded 3D spheroids Basic Protocol 2: Delivery of siRNA and collection of matrix-free 3D spheroids Alternate Protocol 3: Delivery of siRNA and collection of matrix-embedded spheroids Basic Protocol 3: RNA and protein extraction from spheroids for characterization of gene knockdown.

An aptamer-mediated base editing platform for simultaneous knockin and multiple gene knockout for allogeneic CAR-T cells generation.

Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. In conventional gene editing platforms that rely on nuclease activity, such as clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR-Cas9), allow efficient introduction of genetic modifications; however, these modifications occur via the generation of DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations and genotoxicity. Here, we apply a novel modular RNA aptamer-mediated Pin-point base editing platform to simultaneously introduce multiple gene knockouts and site-specific integration of a transgene in human primary T cells. We demonstrate high editing efficiency and purity at all target sites and significantly reduced frequency of chromosomal translocations compared with the conventional CRISPR-Cas9 system. Site-specific knockin of a chimeric antigen receptor and multiplex gene knockout are achieved within a single intervention and without the requirement for additional sequence-targeting components. The ability to perform complex genome editing efficiently and precisely highlights the potential of the Pin-point platform for application in a range of advanced cell therapies.

The timing of antenatal care access for adolescent pregnancies in Cape Town, South Africa.

BACKGROUND:  Late antenatal care (ANC)-seeking among pregnant adolescents threatens their health outcomes, and the health outcomes of their new-borns. South Africa has experienced a rapid increase in adolescent pregnancies during the COVID-19 pandemic, adding to the existing concerns around adolescent pregnancy care-seeking behaviour. AIM:  The main aim of this study was to investigate the causes and covariates of late ANC access among adolescents in the Cape Town Metropole, South Africa. SETTING:  Three public healthcare facilities in the Cape Town Metropole, 2018-2019. METHODS:  A retrospective, cross-sectional study on ANC seeking behaviour was conducted, surveying 202 adolescents. Late attendance was defined as attending ≥ 3 months. For this study, adolescents were defined as women aged 16-18 years. The sample was restricted to adolescents who used public healthcare facilities or who did not attend at all. Data were analysed using univariate, bivariate and multivariate methods. RESULTS:  A total of 50.8% (n = 99/195) of the pregnant adolescents in the sample had their first ANC visit 3 months. 14.9% (n = 29/195) did not attend at all. Major contributors to delayed care-seeking include poor pregnancy identification (n = 45/99, 45.5%), and a lack of information about ANC. Age, education, and alcohol consumption were significant predictors of delayed care-seeking. CONCLUSION:  Delayed ANC attendance contributes to negative long-term health outcomes for pregnant adolescents and their new-borns. Improving access to pregnancy tests in the public sector could benefit adolescents with earlier pregnancy identification. Adolescents need to be made aware of their care seeking options.Contribution: There is evidence of long-term health impacts of late ANC attendance by pregnant adolescents, but there is an absence of evidence on the timing and barriers of late care-seeking behaviour. In this study, late ANC attendance among adolescents was associated with late pregnancy identification and poor knowledge of care options.

A Novel CRISPR Interference Effector Enabling Functional Gene Characterization with Synthetic Guide RNAs.

While CRISPR interference (CRISPRi) systems have been widely implemented in pooled lentiviral screening, there has been limited use with synthetic guide RNAs for the complex phenotypic readouts enabled by experiments in arrayed format. Here we describe a novel deactivated Cas9 fusion protein, dCas9-SALL1-SDS3, which produces greater target gene repression than first or second generation CRISPRi systems when used with chemically modified synthetic single guide RNAs (sgRNAs), while exhibiting high target specificity. We show that dCas9-SALL1-SDS3 interacts with key members of the histone deacetylase and Swi-independent three complexes, which are the endogenous functional effectors of SALL1 and SDS3. Synthetic sgRNAs can also be used with in vitro-transcribed dCas9-SALL1-SDS3 mRNA for short-term delivery into primary cells, including human induced pluripotent stem cells and primary T cells. Finally, we used dCas9-SALL1-SDS3 for functional gene characterization of DNA damage host factors, orthogonally to small interfering RNA, demonstrating the ability of the system to be used in arrayed-format screening.

'The nurse did not even greet me': how informed versus non-informed patients evaluate health systems responsiveness in South Africa.

INTRODUCTION: Universal Health Coverage is not only about access to health services but also about access to high-quality care, since poor experiences may deter patients from accessing care. Evidence shows that quality of care drives health outcomes, yet little is known about non-clinical dimensions of care, and patients' experience thereof relative to satisfaction with visits. This paper investigates the role of non-clinical dimensions of care in patient satisfaction. METHODS: Our study describes the interactions of informed and non-informed patients with primary healthcare workers at 39 public healthcare facilities in two metropolitan centres in two South African provinces. Our analysis included 1357 interactions using standardised patients (for informed patients) and patients' exit interviews (for non-informed patients). The data were combined for three types of visits: contraception, hypertension and tuberculosis. We describe how satisfaction with care was related to patients' experiences of non-clinical dimensions. RESULTS: We show that when real patients (RPs) reported being satisfied (vs dissatisfied) with a visit, it was associated with a 30% increase in the probability that a patient is greeted at the facilities. Likewise, when the RPs reported being satisfied (vs dissatisfied) with the visit, it was correlated with a 15% increase in the prospect that patients are pleased with healthcare workers' explanations of health conditions. CONCLUSION: Informed patients are better equipped to assess health-systems responsiveness in healthcare provision. Insights into responsiveness could guide broader efforts aimed at targeted education and empowerment of primary healthcare users to strengthen health systems and shape expectations for appropriate care and conduct.

CRISPR-mediated transcriptional activation with synthetic guide RNA.

The CRISPR-Cas9 system has been adapted for transcriptional activation (CRISPRa) and several second-generation CRISPRa systems (including VPR, SunTag, and SAM) have been developed to recruit different transcriptional activators to a deactivated Cas9, which is guided to a transcriptional start site via base complementarity with a target guide RNA. Multiple studies have shown the benefit of CRISPRa using plasmid or lentiviral expressed guide RNA, but the use of synthetic guide RNA has not been reported. Here we demonstrate the effective use of synthetic guide RNA for gene activation via CRISPRa. CRISPRa crRNA may be used with a canonical tracrRNA using the VPR or SunTag activation systems or with an extended tracrRNA containing an aptamer sequence for the SAM system. Transcriptional activation with synthetic crRNA:tracrRNA is comparable to activation achieved with expression vectors and combining several crRNA sequences targeting the same gene can enhance transcriptional activation. The use of synthetic crRNA is also ideal for simultaneous activation of multiple genes or use with dCas9-VPR mRNA when viral transduction is not feasible. Here, we perform a proof-of-principle arrayed screen using a CRISPRa crRNA library consisting of 153 cytokine receptor targets to identify regulators of IL-6 cytokine secretion. Together, these results demonstrate the suitability of synthetic CRISPRa guide RNA for high throughput, arrayed screening applications which allow for more complex phenotypic readouts to complement viability and drug resistance assays typically used in a pooled screening format.

ErCas12a CRISPR-MAD7 for Model Generation in Human Cells, Mice, and Rats.

MAD7 is an engineered class 2 type V-A CRISPR-Cas (Cas12a/Cpf1) system isolated from Eubacterium rectale. Analogous to Cas9, it is an RNA-guided nuclease with demonstrated gene editing activity in Escherichia coli and yeast cells. Here, we report that MAD7 is capable of generating indels and fluorescent gene tagging of endogenous genes in human HCT116 and U2OS cancer cell lines, respectively. In addition, MAD7 is highly proficient in generating indels, small DNA insertions (23 bases), and larger integrations ranging from 1 to 14 kb in size in mouse and rat embryos, resulting in live-born transgenic animals. Due to the different protospacer adjacent motif requirement, small-guide RNA, and highly efficient targeted gene disruption and insertions, MAD7 can expand the CRISPR toolbox for genome enginnering across different systems and model organisms.

Use of simulated patients to assess hypertension case management at public healthcare facilities in South Africa.

OBJECTIVE: Our study aims to evaluate hypertensive case management in South Africa's public health sector using simulated patients. METHOD: Our study describes interactions between hypertensive simulated patients and primary healthcare workers at 39 public sector healthcare facilities in two metropolitan centres in the Eastern and Western Cape Provinces of South Africa. Our analysis focus on 97 interactions where our eight simulated patients tested within range for stage 1 hypertension, that is with SBP 140-159 mmHg and/or DBP 90-99 mmHg. For this subset, we describe how healthcare workers communicated the outcome of the blood pressure test, and whether they follow government guidelines on risk assessment and lifestyle advice. RESULTS: Healthcare workers highlighted the risks associated with hypertension in one out of three cases and stressed the importance of regular monitoring of blood pressure in less than half of cases. Hypertensive patients received advice on all six lifestyle risk factors in 8% of cases. 39% of patients received no lifestyle advice at all. In one out of four cases, hypertensive patients left the facility without a hypertension diagnosis and with no prospect of a follow-up visit. CONCLUSION: Simulated patients can assess the quality of hypertension case management, yielding granular and comprehensive information that can help mobilize resources to improve care. The management of hypertension patients in South African public healthcare facilities is critically insufficient. Given that hypertension is responsible for a rising share of deaths in South Africa and many of these deaths are preventable, urgent intervention is needed.

Demand-Side Causes and Covariates of Late Antenatal Care Access in Cape Town, South Africa.

Objectives The objective of this study was to investigate the causes and covariates of late antenatal care access in South Africa. Methods A cross-sectional study was conducted, interviewing 221 women at four public-sector labour wards in Cape Town, South Africa in 2014. A definition of late attendance as attending ≥ 5 months was used. Data were analysed using univariate, bivariate and multivariate methods. Results Of the women who attended antenatal care at a public-sector clinic (n = 213, 96.4%), more than half (51.2%) attended ≥ 3 months and < 5 months, and a quarter (26.3%) attended ≥ 5 months. For those attending ≥ 5 months, 51.8% cited late recognition of pregnancy as the major reason for delayed attendance. Supply-side barriers were not identified as large contributing factors to delayed attendance. Late antenatal care access was predominantly associated with demand-side factors. Women who accessed antenatal care ≥ 5 months were more likely to be in the poorest 40% of the wealth-index distribution (p = 0.034) and to not have completed high school (p = 0.006). They were also more likely to report alcohol consumption during pregnancy (p = 0.020) and be multiparous (p = 0.035). Having completed high school was protective of late antenatal care access in stepwise logistic regression analysis (OR 0.403, CI 0.210-0.773, p < 0.01). For women who attended ≥ 3 months, late access was associated with unwanted pregnancy (p = 0.030). Conclusions for Practice Improved access to pregnancy tests could assist in earlier pregnancy identification. Interventions to increase awareness of the importance of early antenatal care attendance among vulnerable women may help.

The rise and fall of mortality inequality in South Africa in the HIV era.

Post-apartheid South Africa has seen an unprecedented rise and fall of mortality in less than two decades as a result of the HIV/AIDS epidemic and the subsequent rollout of free antiretroviral therapy (ART). Since the incidence of both was not equal for rich and poor, it is likely to also have affected disparities in health and survival chances by income. We use large nationwide surveys for 2001, 2007 and 2011 to obtain estimates of average income and mortality at the aggregate level of a municipality, and then to examine changes in mortality - and in inequality in mortality by income ─ over time. Using concentration indices to measure health inequality, we demonstrate that both the mean mortality level and absolute inequality in mortality by income rose rapidly until 2006, and declined again sharply since the rollout of free ART. Relative inequalities in mortality by income, however, remained fairly stable over the 2001-2011 period. The analysis of age-sex-specific mortality rates shows that it was in particular for adults aged 18-59 years that mortality and absolute inequality increased substantially between 2001 and 2006, followed by a rapid drop thereafter. These trends were far more pronounced for males than females. This means that the HIV/AIDS epidemic has taken a serious death toll, which was concentrated disproportionately among the poorest segments of the population and especially affected (older) males. While South Africa has been very successful in curbing the overall mortality trend since 2006, large disparities in survival prospects by income, race and gender continue to exist. Targeted efforts are required if it wants to further reduce the very unequal chances of living to old age for richer and poorer population groups of all ages.

Measuring Quality Gaps in TB Screening in South Africa Using Standardised Patient Analysis.

This is the first multi-district Standardised Patient (SP) study in South Africa. It measures the quality of TB screening at primary healthcare (PHC) facilities. We hypothesise that TB screening protocols and best practices are poorly adhered to at the PHC level. The SP method allows researchers to observe how healthcare providers identify, test and advise presumptive TB patients, and whether this aligns with clinical protocols and best practice. The study was conducted at PHC facilities in two provinces and 143 interactions at 39 facilities were analysed. Only 43% of interactions resulted in SPs receiving a TB sputum test and being offered an HIV test. TB sputum tests were conducted routinely (84%) while HIV tests were offered less frequently (47%). Nurses frequently neglected to ask SPs whether their household contacts had confirmed TB (54%). Antibiotics were prescribed without taking temperatures in 8% of cases. The importance of returning to the facility to receive TB test results was only explained in 28%. The SP method has highlighted gaps in clinical practice, signalling missed opportunities. Early detection of sub-optimal TB care is instrumental in decreasing TB-related morbidity and mortality. The findings provide the rationale for further quality improvement work in TB management.

Lactobacillus gasseri CRISPR-Cas9 characterization In Vitro reveals a flexible mode of protospacer-adjacent motif recognition.

While the CRISPR-Cas9 system from S. pyogenes is a powerful genome engineering tool, additional programmed nucleases would enable added flexibility in targeting space and multiplexing. Here, we characterized a CRISPR-Cas9 system from L. gasseri and found that it has modest activity in a cell-free lysate assay but no activity in mammalian cells even when altering promoter, position of tag sequences and NLS, and length of crRNA:tracrRNA. In the lysate assay we tested over 400 sequential crRNA target sequences and found that the Lga Cas9 PAM is NNGA/NDRA, different than NTAA predicted from the native bacterial host. In addition, we found multiple instances of consecutive crRNA target sites, indicating flexibility in either PAM sequence or distance from the crRNA target site. This work highlights the need for characterization of new CRISPR systems and highlights the non-triviality of porting them into eukaryotes as gene editing tools.

A Single Amide Linkage in the Passenger Strand Suppresses Its Activity and Enhances Guide Strand Targeting of siRNAs.

Potential in vivo applications of RNA interference (RNAi) require suppression of various off-target activities. Herein, we report that replacement of a single phosphate linkage between the first and second nucleosides of the passenger strand with an amide linkage almost completely abolished its undesired activity and restored the desired activity of guide strands that had been compromised by unfavorable amide modifications. Molecular modeling suggested that the observed effect was most likely due to suppressed loading of the amide-modified strand into Ago2 caused by inability of amide to adopt the conformation required for the backbone twist that docks the first nucleotide of the guide strand in the MID domain of Ago2. Eliminating off-target activity of the passenger strand will be important for improving therapeutic potential of RNAi.

Minimal 2'-O-methyl phosphorothioate linkage modification pattern of synthetic guide RNAs for increased stability and efficient CRISPR-Cas9 gene editing avoiding cellular toxicity.

Since its initial application in mammalian cells, CRISPR-Cas9 has rapidly become a preferred method for genome engineering experiments. The Cas9 nuclease is targeted to genomic DNA using guide RNAs (gRNA), either as the native dual RNA system consisting of a DNA-targeting CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), or as a chimeric single guide RNA (sgRNA). Entirely DNA-free CRISPR-Cas9 systems using either Cas9 protein or Cas9 mRNA and chemically synthesized gRNAs allow for transient expression of CRISPR-Cas9 components, thereby reducing the potential for off-targeting, which is a significant advantage in therapeutic applications. In addition, the use of synthetic gRNA allows for the incorporation of chemical modifications for enhanced properties including improved stability. Previous studies have demonstrated the utility of chemically modified gRNAs, but have focused on one pattern with multiple modifications in co-electroporation with Cas9 mRNA or multiple modifications and patterns with Cas9 plasmid lipid co-transfections. Here we present gene editing results using a series of chemically modified synthetic sgRNA molecules and chemically modified crRNA:tracrRNA molecules in both electroporation and lipid transfection assessing indel formation and/or phenotypic gene knockout. We show that while modifications are required for co-electroporation with Cas9 mRNA, some modification patterns of the gRNA are toxic to cells compared to the unmodified gRNA and most modification patterns do not significantly improve gene editing efficiency. We also present modification patterns of the gRNA that can modestly improve Cas9 gene editing efficiency when co-transfected with Cas9 mRNA or Cas9 protein (> 1.5-fold difference). These results indicate that for certain applications, including those relevant to primary cells, the incorporation of some, but not all chemical modification patterns on synthetic crRNA:tracrRNA or sgRNA can be beneficial to CRISPR-Cas9 gene editing.

A comparable yardstick: adjusting for education bias in South African health system responsiveness ratings.

Health-system responsiveness (HSR) measures the experience of health-system users in terms of the non-clinical aspects of the health system. This has been operationalized as a measurable construct in multiple surveys and studies. According to the literature, reporting behaviour may vary systematically across socio-demographic characteristics. In this study we explore the association between education levels and reporting behaviour in terms of HSR in South Africa using data from the World Health Organization Study on Global Ageing and Adult Health for South Africa (WHO SAGE) conducted in 2007 and 2008. We consider the reporting behaviour of 1499 adults aged 50 and older in terms of the reported HSR for their most recent outpatient provider visit during the preceding 12 months. More specifically, we explore whether there are systematic biases in reporting behaviour by education levels and other socio-economic covariates through the use of data from anchoring vignettes. These are questions depicting hypothetical HSR scenarios which provide a fixed benchmark for comparing individuals' own HSR ratings and identifying potential reporting biases. Using a hierarchical-ordered probit model in regression analysis, we found large differences in HSR ratings between the lowest and highest education groups after adjusting for reporting bias using the anchoring vignettes. This finding holds across all seven HSR domains captured in the WHO SAGE dataset. In the most extreme case, individuals with no education are likely to underreport poor HSR by between 2.6 and 9.4% percentage points compared with individuals with secondary schooling or higher. Policy-makers need to take cognizance of potential reporting biases in HSR ratings and make the necessary adjustments to obtain data that are as true and accurate as possible. The need for this is especially acute in a country such as South Africa with large socio-economic inequalities and disparities in access to healthcare.

Amide linkages mimic phosphates in RNA interactions with proteins and are well tolerated in the guide strand of short interfering RNAs.

While the use of RNA interference (RNAi) in molecular biology and functional genomics is a well-established technology, in vivo applications of synthetic short interfering RNAs (siRNAs) require chemical modifications. We recently found that amides as non-ionic replacements for phosphodiesters may be useful modifications for optimization of siRNAs. Herein, we report a comprehensive study of systematic replacement of a single phosphate with an amide linkage throughout the guide strand of siRNAs. The results show that amides are surprisingly well tolerated in the seed and central regions of the guide strand and increase the silencing activity when placed between nucleosides 10 and 12, at the catalytic site of Argonaute. A potential explanation is provided by the first crystal structure of an amide-modified RNA-DNA with Bacillus halodurans RNase H1. The structure reveals how small changes in both RNA and protein conformation allow the amide to establish hydrogen bonding interactions with the protein. Molecular dynamics simulations suggest that these alternative binding modes may compensate for interactions lost due to the absence of a phosphodiester moiety. Our results suggest that an amide can mimic important hydrogen bonding interactions with proteins required for RNAi activity and may be a promising modification for optimization of biological properties of siRNAs.

High-content analysis screening for cell cycle regulators using arrayed synthetic crRNA libraries.

The CRISPR-Cas9 system has been utilized for large-scale, loss-of-function screens mainly using lentiviral pooled formats and cell-survival phenotypic assays. Screening in an arrayed format expands the types of phenotypic readouts that can be used to now include high-content, morphology-based assays, and with the recent availability of synthetic crRNA libraries, new studies are emerging. Here, we use a cell cycle reporter cell line to perform an arrayed, synthetic crRNA:tracrRNA screen targeting 169 genes (>600 crRNAs) and used high content analysis (HCA) to identify genes that regulate the cell cycle. Seven parameters were used to classify cells into cell cycle categories and multiple parameters were combined using a new analysis technique to identify hits. Comprehensive hit follow-up experiments included target gene expression analysis, confirmation of DNA insertions/deletions, and validation with orthogonal reagents. Our results show that most hits had three or more independent crRNAs per gene that demonstrated a phenotype with consistent individual parameters, indicating that our screen produced high-confidence hits with low off-target effects and allowed us to identify hits with more subtle phenotypes. The results of our screen demonstrate the power of using arrayed, synthetic crRNAs for functional phenotypic screening using multiparameter HCA assays.

Versatility of chemically synthesized guide RNAs for CRISPR-Cas9 genome editing.

The CRISPR-Cas9 system has become the most popular and efficient method for genome engineering in mammalian cells. The Streptococcus pyogenes Cas9 nuclease can function with two types of guide RNAs: the native dual crRNA and tracrRNA (crRNA:tracrRNA) or a chimeric single guide RNA (sgRNA). Although sgRNAs expressed from a DNA vector are predominant in the literature, guide RNAs can be rapidly generated by chemical synthesis and provide equivalent functionality in gene editing experiments. This review highlights the attributes and advantages of chemically synthesized guide RNAs including the incorporation of chemical modifications to enhance gene editing efficiencies in certain applications. The use of synthetic guide RNAs is also uniquely suited to genome-scale high throughput arrayed screening, particularly when using complex phenotypic assays for functional genomics studies. Finally, the use of synthetic guide RNAs along with DNA-free sources of Cas9 (mRNA or protein) allows for transient CRISPR-Cas9 presence in the cell, thereby resulting in a decreased probability of off-target events.

Conjugation and Evaluation of Triazole-Linked Single Guide RNA for CRISPR-Cas9 Gene Editing.

The CRISPR-Cas9 gene editing system requires Cas9 endonuclease and guide RNAs (either the natural dual RNA consisting of crRNA and tracrRNA or a chimeric single guide RNA) that direct site-specific double-stranded DNA cleavage. This communication describes a click ligation approach that uses alkyne-azide cycloaddition to generate a triazole-linked single guide RNA (sgRNA). The conjugated sgRNA shows efficient and comparable genome editing activity to natural dual RNA and unmodified sgRNA constructs.