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In case-control single-cell RNA-seq studies, sample-level labels are transferred onto individual cells, labeling all case cells as affected, when in reality only a small fraction of them may actually be perturbed. Here, using simulations, we demonstrate that the standard approach to single cell analysis fails to isolate the subset of affected case cells and their markers when either the affected subset is small, or when the strength of the perturbation is mild. To address this fundamental limitation, we introduce HiDDEN, a computational method that refines the case-control labels to accurately reflect the perturbation status of each cell. We show HiDDEN's superior ability to recover biological signals missed by the standard analysis workflow in simulated ground truth datasets of cell type mixtures. When applied to a dataset of human multiple myeloma precursor conditions, HiDDEN recapitulates the expert manual annotation and discovers malignancy in early stage samples missed in the original analysis. When applied to a mouse model of demyelination, HiDDEN identifies an endothelial subpopulation playing a role in early stage blood-brain barrier dysfunction. We anticipate that HiDDEN should find wide usage in contexts that require the detection of subtle transcriptional changes in cell types across conditions., (© 2024. The Author(s).)

Tuberculosis (TB) is a chronic condition that weakens the immune system, causes structural changes in the lungs, and can lead to infections by other bacterial pathogens. Very few studies have been done to understand the magnitude of co-infection with other bacterial pathogens, so this study was conducted to understand the co-infection pattern and burden. A total of 174 microbiologically confirmed pulmonary TB patients' samples, identified by cartridge-based nucleic acid amplification test, were further tested for other bacterial pathogens by culture over a period of five months from May 2023 to September 2023. The isolates' identification and drug susceptibility were performed using the VITEK 2 system (bioMérieux, Marcy-l'Étoile, France). Of the 174 pulmonary samples tested, 19 samples grew a significant amount of other bacterial pathogens, making the prevalence 10.91% (19/174). Among the pulmonary samples tested, 54.59% were sputum, 38.5% were bronchoalveolar lavage, and 6.89% were endotracheal aspirate. Additionally, 70.11% of the patients tested were in the age group of 19-60 years. Of the patients who had co-infection, 94.73% (18/19) were male. The most common bacterial infection was caused by Pseudomonas aeruginosa , which was identified in 36.84% of the co-infection cases (7/19). This was followed by Acinetobacter baumannii in 31.57% (6/19), Klebsiella pneumoniae in 26.31% (5/19), and Stenotrophomonas maltophilia in 5.28% (1/19). Acinetobacter baumannii and Klebsiella pneumoniae showed high drug resistance, ranging from 60% to 100% against various groups of drugs tested. None of the patient samples with co-infection showed rifampicin resistance. Among all the samples with co-infection, the majority (42.10%, or 8/19) had a high load of Mycobacterium tuberculosis complex detected by CBNAAT Ultra (Cepheid, Sunnyvale, California). Pseudomonas aeruginosa , Acinetobacter baumannii , and Klebsiella pneumoniae are unusual pathogens causing infection in community patients and are known to cause illness in hospitalized patients. These organisms' resistance was also similar to the resistance shown by hospital-acquired infections. This indicates that bacterial co-infection in pulmonary TB patients will be similar to the pattern of hospital-acquired infections. The high prevalence of bacterial co-infections (10.91%) in patients with pulmonary TB poses a significant challenge as these bacterial pathogens are not susceptible to anti-tubercular drugs. Therefore, comprehensive screening for other bacterial infections in all pulmonary TB patients is crucial for effective treatment and outcomes., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. Institutional Ethics Committee of ESIC MC&H, Faridabad issued approval 134 X/11/13/2024- IEC/DHR/04. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Bir et al.)

Drug enantiomers can possess vastly different pharmacological properties, yet they are identical in their chemical composition and structural connectivity. Thus, resolving enantiomers poses a great challenge in the field of separation science. Enantiomer separations necessitate interaction of the analyte with a chiral environment─in mass spectrometry-based analysis, a common approach is through a three-point interaction with a chiral selector commonly introduced during sample preparation. In select cases, the structural difference imparted through noncovalent complexation results in enantiomer-specific structural differences, facilitating measurement using a structurally selective analytical technique such as ion mobility-mass spectrometry (IM-MS). In this work, we investigate the direct IM-MS differentiation of chiral drug compounds using mononuclear copper complexes incorporating an amino acid chiral selector. A panel of 20 chiral drugs and drug-like compounds were investigated for separation, and four l-amino acids (l-histidine, l-tryptophan, l-proline, and l-tyrosine) were evaluated as chiral selectors (CS) to provide the chiral environment necessary for differentiation. Enantiomer differentiation was achieved for four chiral molecule pairs ( R / S -thalidomide, R / S -baclofen, R / S -metoprolol, and d/l-panthenol) with two-peak resolution ( R p-p ) values ranging from 0.7 (>10% valley) to 1.5 (baseline separation). Calibration curves relating IM peak areas to enantiomeric concentrations enabled enantiomeric excess quantitation of racemic thalidomide and metoprolol with residuals of 5.7 and 2.5%, respectively. Theoretical models suggest that Cu II and l-histidine complexation around the analyte chiral center is important for gas-phase stereoselectivity. This study demonstrates the potential of combining enantioselective noncovalent copper complexation with structurally selective IM-MS for differentiating chiral drugs and drug-like compounds.

We report a rare case of splenic tuberculosis (TB) in a male patient with a competent immune system who had no previous record of pulmonary TB. A 56-year-old male patient came to our outpatient department complaining of upper abdominal pain with a few episodes of vomiting for three days. He had alcoholism, smoked for 15 years, and had no past history of diabetes mellitus, hypertension, TB, or HIV. An abdominal ultrasound and CT scan at admission showed pancreatitis with a splenic abscess. After five days of admission, the patient's vitals deteriorated, and he had severe abdominal pain. CT scan suggested a splenic abscess rupture with hemoperitoneum. An emergency exploratory laparotomy was performed, and a splenectomy was done due to the splenic abscess rupture. A cartridge-based nucleic acid amplification test from splenic intracapsular fluid detected a trace Mycobacterium tuberculosis complex. The patient was discharged after starting first-line antitubercular treatment for six months. After three months of follow-up, the patient was doing well with no complaints., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, Ranjan et al.)

Endothelial cells (ECs) mediate several biological functions that are relevant to atherosclerosis and coronary artery disease (CAD), regulating an array of vital processes including vascular tone, wound healing, reactive oxygen species, shear stress response, and inflammation. Although which of these functions is linked causally with CAD development and/or progression is not yet known, genome-wide association studies have implicated more than 400 loci associated with CAD risk, among which several have shown EC-relevant functions. Given the arduous process of mechanistically interrogating single loci to CAD, high-throughput variant characterization methods, including pooled Clustered Regularly Interspaced Short Palindromic Repeats screens, offer exciting potential to rapidly accelerate the discovery of bona fide EC-relevant genetic loci. These discoveries in turn will broaden the therapeutic avenues for CAD beyond lipid lowering and behavioral risk modification to include EC-centric modalities of risk prevention and treatment., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Linking variants from genome-wide association studies (GWAS) to underlying mechanisms of disease remains a challenge 1-3 . For some diseases, a successful strategy has been to look for cases in which multiple GWAS loci contain genes that act in the same biological pathway 1-6 . However, our knowledge of which genes act in which pathways is incomplete, particularly for cell-type-specific pathways or understudied genes. Here we introduce a method to connect GWAS variants to functions. This method links variants to genes using epigenomics data, links genes to pathways de novo using Perturb-seq and integrates these data to identify convergence of GWAS loci onto pathways. We apply this approach to study the role of endothelial cells in genetic risk for coronary artery disease (CAD), and discover 43 CAD GWAS signals that converge on the cerebral cavernous malformation (CCM) signalling pathway. Two regulators of this pathway, CCM2 and TLNRD1, are each linked to a CAD risk variant, regulate other CAD risk genes and affect atheroprotective processes in endothelial cells. These results suggest a model whereby CAD risk is driven in part by the convergence of causal genes onto a particular transcriptional pathway in endothelial cells. They highlight shared genes between common and rare vascular diseases (CAD and CCM), and identify TLNRD1 as a new, previously uncharacterized member of the CCM signalling pathway. This approach will be widely useful for linking variants to functions for other common polygenic diseases., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Population-based association studies have identified many genetic risk loci for coronary artery disease (CAD), but it is often unclear how genes within these loci are linked to CAD. Here, we perform interaction proteomics for 11 CAD-risk genes to map their protein-protein interactions (PPIs) in human vascular cells and elucidate their roles in CAD. The resulting PPI networks contain interactions that are outside of known biology in the vasculature and are enriched for genes involved in immunity-related and arterial-wall-specific mechanisms. Several PPI networks derived from smooth muscle cells are significantly enriched for genetic variants associated with CAD and related vascular phenotypes. Furthermore, the networks identify 61 genes that are found in genetic loci associated with risk of CAD, prioritizing them as the causal candidates within these loci. These findings indicate that the PPI networks we have generated are a rich resource for guiding future research into the molecular pathogenesis of CAD., (© 2024. The Author(s).)

Genome-wide association studies (GWASs) are a valuable tool for understanding the biology of complex human traits and diseases, but associated variants rarely point directly to causal genes. In the present study, we introduce a new method, polygenic priority score (PoPS), that learns trait-relevant gene features, such as cell-type-specific expression, to prioritize genes at GWAS loci. Using a large evaluation set of genes with fine-mapped coding variants, we show that PoPS and the closest gene individually outperform other gene prioritization methods, but observe the best overall performance by combining PoPS with orthogonal methods. Using this combined approach, we prioritize 10,642 unique gene-trait pairs across 113 complex traits and diseases with high precision, finding not only well-established gene-trait relationships but nominating new genes at unresolved loci, such as LGR4 for estimated glomerular filtration rate and CCR7 for deep vein thrombosis. Overall, we demonstrate that PoPS provides a powerful addition to the gene prioritization toolbox., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Preeclampsia and gestational hypertension are common pregnancy complications associated with adverse maternal and child outcomes. Current tools for prediction, prevention and treatment are limited. Here we tested the association of maternal DNA sequence variants with preeclampsia in 20,064 cases and 703,117 control individuals and with gestational hypertension in 11,027 cases and 412,788 control individuals across discovery and follow-up cohorts using multi-ancestry meta-analysis. Altogether, we identified 18 independent loci associated with preeclampsia/eclampsia and/or gestational hypertension, 12 of which are new (for example, MTHFR-CLCN6, WNT3A, NPR3, PGR and RGL3), including two loci (PLCE1 and FURIN) identified in the multitrait analysis. Identified loci highlight the role of natriuretic peptide signaling, angiogenesis, renal glomerular function, trophoblast development and immune dysregulation. We derived genome-wide polygenic risk scores that predicted preeclampsia/eclampsia and gestational hypertension in external cohorts, independent of clinical risk factors, and reclassified eligibility for low-dose aspirin to prevent preeclampsia. Collectively, these findings provide mechanistic insights into the hypertensive disorders of pregnancy and have the potential to advance pregnancy risk stratification., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Carbapenemase producing gram-negative bacteria (GNB) has become a huge problem in majority of tertiary care centers worldwide. They are associated with very high morbidity and mortality rates, especially when they cause invasive infections. Therefore, rapid detection of these organisms is very important for prompt and adequate antibiotic therapy as well as infection control. The aim of this study was rapid detection of carbapenemase genes and thereby likely carbapenem resistance, 24-48 hours in advance, directly from the positive-flagged blood culture bottles using CHROMagar and Xpert® Carba-R., Methods: Aspirate from positively flagged blood culture bottles was subjected to differential centrifuge. All gram-negative bacilli on gram stain from the deposit were processed in Xpert® Carba-R and inoculated on CHROMagar. The presence of genes and growth on CHROMagar was compared with carbapenem resistance on VITEK-2 Compact., Results: A total of 119 GNB isolates were processed. One or more of the carbapenemase genes were detected in 80 isolates. On comparison with VITEK-2 result, 92 samples showed concordance for carbapenem resistance 48 hours in advance. There was discordance in 21 isolates with 12 major errors and 09 minor errors. The sensitivity of direct Xpert® Carba-R test for rapid detection of carbapenem resistance, 48 hours in advance, was 81.42%. The sensitivity of direct CHROMagar test for accurate detection of carbapenem resistance, 24 hours in advance, was 92.06%., Conclusion: The ability to detect carbapenem resistance with very high accuracy, 48 hours in advance, helps in appropriate antibiotic therapy and implementation of effective infection control practices., Competing Interests: The authors have none to declare., (© 2021 Director General, Armed Forces Medical Services. Published by Elsevier, a division of RELX India Pvt. Ltd.)

Endothelial cells (EC) are an important mediator of atherosclerosis and vascular disease. Their exposure to atherogenic risk factors such as hypertension and serum cholesterol leads to endothelial dysfunction and many disease-associated processes. Identifying which of these multiple EC functions is causally related to disease risk has been challenging. There is evidence from in vivo models and human sequencing studies that dysregulation of nitric oxide production directly affects risk of coronary artery disease. Human genetics can help prioritize the other EC functions with causal relationships because germline mutations are acquired at birth and serve as a randomized test of which pathways affect disease risk. Though several coronary artery disease risk variants have been linked to EC function, this process has been slow and laborious. Unbiased analyses of EC dysfunction using multiomic approaches promise to identify the causal genetic mechanisms responsible for vascular disease. Here, we review the data from genomic, epigenomic, and transcriptomic studies that prioritize EC-specific causal pathways. New methods that CRISPR (clustered regularly interspaced short palindromic repeats) perturbation technology with genomic, epigenomic, and transcriptomic analysis promise to speed up the characterization of disease-associated genetic variation. We summarize several recent studies in ECs which use high-throughput genetic perturbation to identify disease-relevant pathways and novel mechanisms of disease. These genetically validated pathways can accelerate the identification of drug targets for the prevention and treatment of atherosclerosis.

The discovery of genetic loci associated with complex diseases has outpaced the elucidation of mechanisms of disease pathogenesis. Here we conducted a genome-wide association study (GWAS) for coronary artery disease (CAD) comprising 181,522 cases among 1,165,690 participants of predominantly European ancestry. We detected 241 associations, including 30 new loci. Cross-ancestry meta-analysis with a Japanese GWAS yielded 38 additional new loci. We prioritized likely causal variants using functionally informed fine-mapping, yielding 42 associations with less than five variants in the 95% credible set. Similarity-based clustering suggested roles for early developmental processes, cell cycle signaling and vascular cell migration and proliferation in the pathogenesis of CAD. We prioritized 220 candidate causal genes, combining eight complementary approaches, including 123 supported by three or more approaches. Using CRISPR-Cas9, we experimentally validated the effect of an enhancer in MYO9B, which appears to mediate CAD risk by regulating vascular cell motility. Our analysis identifies and systematically characterizes >250 risk loci for CAD to inform experimental interrogation of putative causal mechanisms for CAD., (© 2022. The Author(s).)

Background: All four dengue serotypes cause infection, with one of them predominantly reported from a particular geographical region. Coinfection by more than one serotype is reported from hyperendemic regions. These coinfections are clinically more severe than infection with a single serotype. This study was carried out to detect the predominant dengue serotype and presence of coinfections., Methods: Acute-phase serum samples of patients suffering from dengue infection were collected. They were screened for the presence of IgM, IgG and NS1Ag by a rapid test. Conventional multiplex reverse transcriptase polymerase chain reaction (RT-PCR) and multiplex real-time RT-PCR assays were carried out for detection and serotyping of the dengue virus., Results: A total of 196 samples were positive by the rapid card test. Of these, 139 were NS1Ag positive, 40 were positive only for IgM, 5 were positive only for IgG and 12 samples were positive for different combinations of antigen and antibodies. All four serotypes were detected in these samples by PCR. DENV-3 was found to be most common circulating serotype. A total of 22 cases were found to have coinfection with more than one dengue serotypes. Samples having only antibodies and no antigen on rapid card test were also positive for virus by PCR., Conclusion: Prevalence of dengue co-infections is increasing. Moreover, it is important to screen for dengue virus in those samples also which do not show NS1Ag on rapid tests and have either one or both the antibodies. Real-time multiplex RT-PCR is found to be more sensitive in detecting coinfection than conventional multiplex RT-PCR., Competing Interests: The authors have none to declare., (© 2021 Director General, Armed Forces Medical Services. Published by Elsevier, a division of RELX India Pvt. Ltd.)

Background: Early case detection is a crucial step in the control of tuberculosis (TB). Sputum smear microscopy is the primary method of TB diagnosis in developing countries. The modified Petroff's method using sodium hydroxide at concentrations ranging between 2% and 4% to digest the specimen is widely used in developing countries. A novel ReaSLR (ReaMetrix's Sputum Liquefying Reagent) methodology has been proposed as a simple, easy, low-cost, and better alternative to conventional methods for sputum processing. This study was undertaken to evaluate the performance of the ReaSLR method of sputum processing in comparison with that of the modified Petroff's method., Methods: Early-morning sputum samples were collected. After preparing a direct smear, each sample was divided into two equal halves and processed by both the methods, i.e., modified Petroff's method and ReaSLR method. Direct smears were graded according to Revised National Tuberculosis Control Program grading, and smears prepared after processing by the two different methods were graded according to Center for Disease Control and Prevention grading. Smear microscopy results were compared taking culture results of samples processed by the modified Petroff's method as the gold standard., Results: The rate of smear positivity with the modified Petroff's method (22.22%) was found to be higher than that with direct smear microscopy (13.56%; p = 0.0002) and the ReaSLR method (17.32%; p = 0.04). The modified Petroff's method was found to be 26.76% more sensitive than direct microscopy and 15.59% more sensitive than the ReaSLR method., Conclusion: The ReaSLR method was not superior to the modified Petroff's method for smear microscopy. Although this method was more sensitive than the direct method in smear microscopy, the modified Petroff's method performed much better than the ReaSLR method., Competing Interests: The authors have none to declare., (© 2020 Director General, Armed Forces Medical Services. Published by Elsevier, a division of RELX India Pvt. Ltd.)

Background and Aims: Chronic vascular endothelial inflammation predisposes to atherosclerosis; however, the cell-autonomous roles for endothelial-expressing microRNAs (miRNAs) are poorly understood in this process. MiR-181b is expressed in several cellular constituents relevant to lesion formation. The aim of this study is to examine the role of genetic deficiency of the miR-181b locus in endothelial cells during atherogenesis., Methods and Results: Using a proprotein convertase subtilisin/kexin type 9 (PCSK9)-induced atherosclerosis mouse model, we demonstrated that endothelial cell (EC)-specific deletion of miR-181a2b2 significantly promoted atherosclerotic lesion formation, cell adhesion molecule expression, and the influx of lesional macrophages in the vessel wall. Yet, endothelium deletion of miR-181a2b2 did not affect body weight, lipid metabolism, anti-inflammatory Ly6C low or the pro-inflammatory Ly6C interm and Ly6C high fractions in circulating peripheral blood mononuclear cells (PBMCs), and pro-inflammatory or anti-inflammatory mediators in both bone marrow (BM) and PBMCs. Mechanistically, bulk RNA-seq and gene set enrichment analysis of ECs enriched from the aortic arch intima, as well as single cell RNA-seq from atherosclerotic lesions, revealed that endothelial miR-181a2b2 serves as a critical regulatory hub in controlling endothelial inflammation, cell adhesion, cell cycle, and immune response during atherosclerosis., Conclusions: Our study establishes that deficiency of a miRNA specifically in the vascular endothelium is sufficient to profoundly impact atherogenesis. Endothelial miR-181a2b2 deficiency regulates multiple key pathways related to endothelial inflammation, cell adhesion, cell cycle, and immune response involved in the development of atherosclerosis., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Device-associated infections (DAIs) such as ventilator associated pneumonia (VAP), central line-associated blood stream infection (CLABSI), and catheter-related urinary tract infection (CAUTI) are principal contributors to health hazard and a major preventable threat to patient safety. Robust surveillance of DAI delineates infections, pathogens, resistograms, and facilitates antimicrobial therapy, infection-control, antimicrobial stewardship, and improvement in quality of care., Methods: This prospective outcome surveillance study was conducted amongst 2067 ICU patients in a 1000-bedded teaching hospital. Clinical, laboratory, and environmental surveillance, as well as screening of health care professionals (HCPs) were conducted using the modified US Centers for Disease Control and Prevention-National Healthcare Safety Network definitions and methods. Morbidity, mortality, and health-care indices were analyzed and two-tier infection prevention and control was promulgated., Results: Mean occupancy was 95.34% for 2061 patients of 7381 patients/bed/ICU days. One hundred seventeen episodes of DAI occurred in 1258 patients of 12,882 device-days with mean device utilization ratio of 1.79. Mean rate of DAI was 7.40 per 1000 device days. Multiresistant Pseudomonas aeruginosa was most commonly followed by Acinetobacter. Mean all-cause mortality in ICU was 24.85%, whereas all-cause mortality after DAI was 9.79%. Methicillin-resistant Staphylococcus aureus prevalence was 38.46% amongst health-care professionals., Conclusion: Mean rates of VAP, CLABSI, and CAUTI were 20.69, 2.53, and 2.23 per 1000 device days comparable with Indian and global ICUs. Resolute conviction and sustained momentum in infection prevention and control is an essential step toward patient safety., Competing Interests: The authors have none to declare., (© 2022 Director General, Armed Forces Medical Services. Published by Elsevier, a division of RELX India Pvt. Ltd.)

Background: Hepatitis B (HepB) is an important vaccine preventable infection among healthcare workers (HCWs). Vaccination against Hep B virus, remains the foremost preventive approach. This study aims to measure the antibody titres to Hep B surface antigen (anti-HBs) in a mixed cohort of HCWs. It also aims to study the association between time since vaccination and the anti-HBs titres thus evaluating the duration of seroprotection., Methods: A total of 200 HCWs, including nursing students (n = 112), nursing staff (n = 49), laboratory technicians (n = 30) and doctors (n = 9) who had received all three doses of the Hep B vaccine and met the inclusion criteria of having taken all three doses of vaccine were included in this study. Anti-HBs titres were estimated by bioMérieux mini VIDAS® automated immunoassay based on the principle of enzyme-linked fluorescence assay., Results: Two hundred subjects aged 19 to 52 years were included in the study; mean age was 27.29 ± 0.568 years. Duration since vaccination in the study cohort was ≤ 5 years in 149 (74.5.0%), 6-10 years in 20 (10.0%) and >10 years in 31 (15.5%) subjects. Postvaccination antibody titres were > 100 mIU/ml in 85.0%, 10-100 mIU/ml in 11.0% and ≤ 10 mIU/ml in 3.5%. There was a decline noted in antibody titres as duration after vaccination increased. Increasing age was associated with falling protective titres., Conclusion: The study revealed that majority of the HCWs had adequate anti-HBs titres and were protected after vaccination., Competing Interests: The authors have none to declare., (© 2022 Director General, Armed Forces Medical Services. Published by Elsevier, a division of RELX India Pvt. Ltd.)

Enlargement or aneurysm of the aorta predisposes to dissection, an important cause of sudden death. We trained a deep learning model to evaluate the dimensions of the ascending and descending thoracic aorta in 4.6 million cardiac magnetic resonance images from the UK Biobank. We then conducted genome-wide association studies in 39,688 individuals, identifying 82 loci associated with ascending and 47 with descending thoracic aortic diameter, of which 14 loci overlapped. Transcriptome-wide analyses, rare-variant burden tests and human aortic single nucleus RNA sequencing prioritized genes including SVIL, which was strongly associated with descending aortic diameter. A polygenic score for ascending aortic diameter was associated with thoracic aortic aneurysm in 385,621 UK Biobank participants (hazard ratio = 1.43 per s.d., confidence interval 1.32-1.54, P = 3.3 × 10 -20 ). Our results illustrate the potential for rapidly defining quantitative traits with deep learning, an approach that can be broadly applied to biomedical images., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Genome editing of primary human cells with CRISPR-Cas9 is a powerful tool to study gene function. For many cell types, there are efficient protocols for editing with optimized plasmids for Cas9 and sgRNA expression. Vascular cells, however, remain refractory to plasmid-based delivery of CRISPR machinery for in vitro genome editing due to low transfection efficiency, poor expression of the Cas9 machinery, and toxic effects of the selection antibiotics. Here, we describe a method for high-efficiency editing of primary human vascular cells in vitro using nucleofection for direct delivery of sgRNA:Cas9-NLS ribonucleoprotein complexes. This method is more rapid and its high editing efficiency eliminates the need for additional selection steps. The edited cells can be employed in diverse applications, such as gene expression measurement or functional assays to assess various genetic perturbation effects in vitro. This method proves effective in vascular cells that are refractory to standard genome manipulation techniques using viral plasmid delivery. We anticipate that this technique will be applied to other non-vascular cell types that face similar barriers to efficient genome editing. © 2021 Wiley Periodicals LLC. Basic Protocol: CRISPR-Cas9 genome editing of primary human vascular cells in vitro., (© 2021 Wiley Periodicals LLC.)