Compact CRISPR genetic screens enabled by improved guide RNA library cloning

Genome biology, Journal Year: 2024, Volume and Issue: 25(1)

Published: Jan. 19, 2024

Abstract CRISPR genome editing approaches theoretically enable researchers to define the function of each human gene in specific cell types, but challenges remain efficiently perform genetic perturbations relevant models. In this work, we develop a library cloning protocol that increases sgRNA uniformity and greatly reduces bias existing genome-wide libraries. We demonstrate our libraries can achieve equivalent or better statistical power compared previously reported screens using an order magnitude fewer cells. This improved enables genome-scale technically challenging models screen formats.

Language: Английский

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Human pluripotent stem cell–derived models and drug screening in CNS precision medicine

Annals of the New York Academy of Sciences, Journal Year: 2019, Volume and Issue: 1471(1), P. 18 - 56

Published: March 15, 2019

Abstract Development of effective therapeutics for neurological disorders has historically been challenging partly because lack accurate model systems in which to investigate disease etiology and test new at the preclinical stage. Human stem cells, particularly patient‐derived induced pluripotent cells (iPSCs) upon differentiation, have ability recapitulate aspects pathophysiology are increasingly recognized as robust scalable drug discovery. We review advances deriving cellular models human central nervous system (CNS) using iPSCs along with strategies investigating disease‐relevant phenotypes, translatable biomarkers, therapeutic targets. Given their potential identify novel targets leads, we focus on phenotype‐based, small‐molecule screens employing cell–derived models. Integrated efforts assemble patient iPSC‐derived cell deeply annotated clinicopathological data, molecular drug‐response signatures, may aid stratification patients, diagnostics, clinical trial success, shifting translational science precision medicine approaches. A number remaining challenges, including optimization cost‐effective, large‐scale culture types, incorporation aging into neuronal models, well robustness automation phenotypic assays support quantitative efficacy, toxicity, metabolism testing workflows, covered. Continued advancement field is expected help fully humanize process CNS

Language: Английский

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Recommendations, guidelines, and best practice for the use of human induced pluripotent stem cells for neuropharmacological studies of neuropsychiatric disorders

Neuroscience Applied, Journal Year: 2023, Volume and Issue: 2, P. 101125 - 101125

Published: Jan. 1, 2023

The number of individuals suffering from neuropsychiatric disorders (NPDs) has increased worldwide, with 3 million disability-adjusted life-years calculated in 2019. Though research using various approaches including genetics, imaging, clinical and animal models advanced our knowledge regarding NPDs, we still lack basic the underlying pathophysiological mechanisms. Moreover, there is an urgent need for highly effective therapeutics NPDs. Human induced pluripotent stem cells (hiPSCs) generated somatic enabled scientists to create brain a patient-specific manner. However, are challenges use hiPSCs that be addressed. In current paper, consideration best practices neuropharmacological will discussed. Specifically, provide recommendations practice patient recruitment, collecting demographic, clinical, medical (before after treatment response), diagnostic (including scales) genetic data donors. We highlight considerations donor genetics sex, addition discussing biological technical replicates. Furthermore, present views on selecting control groups/lines, experimental designs, conducting studies hiPSC-based context doing so, explore key issues field concerning reproducibility, statistical analysis, how translate vitro into clinically relevant observations. aim this article resource hiPSC researchers perform robust reproducible studies, ultimate improving identification translation novel therapeutic drugs

Language: Английский

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CRISPR-Cas9 correction of a nonsense mutation in LCA5 rescues lebercilin expression and localization in human retinal organoids

Molecular Therapy — Methods & Clinical Development, Journal Year: 2023, Volume and Issue: 29, P. 522 - 531

Published: May 17, 2023

Mutations in the lebercilin-encoding gene

Language: Английский

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Between and within laboratory reliability of mouse behaviour recorded in home-cage and open-field

Journal of Neuroscience Methods, Journal Year: 2017, Volume and Issue: 300, P. 10 - 19

Published: Dec. 9, 2017

Language: Английский

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Editing the Genome of Human Induced Pluripotent Stem Cells Using CRISPR/Cas9 Ribonucleoprotein Complexes

Methods in molecular biology, Journal Year: 2019, Volume and Issue: unknown, P. 153 - 183

Published: Jan. 1, 2019

Language: Английский

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Considerations for an In Vitro, Cell-Based Testing Platform for Detection of Drug-Induced Inotropic Effects in Early Drug Development. Part 2: Designing and Fabricating Microsystems for Assaying Cardiac Contractility With Physiological Relevance Using Human iPSC-Cardiomyocytes

Frontiers in Pharmacology, Journal Year: 2019, Volume and Issue: 10

Published: Aug. 29, 2019

Contractility of the myocardium engines pumping function heart and is enabled by collective contractile activity its muscle cells: cardiomyocytes. The effects drugs on contractility human cardiomyocytes in vitro can provide mechanistic insight that support prediction clinical cardiac drug early development. Cardiomyocytes differentiated from induced pluripotent stem cells have high potential for overcoming current limitations assays because they attach easily to extracellular materials last long culture, while having human- patient-specific properties. Under these conditions, measurements be non-destructive minimally-invasive, which allow assaying sub-chronic drugs. For this purpose, must reflect physiological settings, not observed cultured derived fetal-like properties their machinery. Primary or tissues origin fully represent cellular properties, but are available, do culture force sensors mechanical actuators. Microengineered systems with a more mature been developed 5 years overcome limitation cell cardiomyocytes, simultaneously measuring endpoints integrated sensors/actuators image-based techniques. Known engineered microenvironments maturity cardiomyocyte also discovered development systems. Based discoveries, here we review design criteria microengineered platforms higher relevance. These involve use electromechanical, chemical morphological cues, co-culture different types three-dimensional microenvironments. We further discuss challenges developing improving novel technologies predicting based form cells. Future research should establish contexts

Language: Английский

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Genome-Wide Association Studies of Endometrial Cancer: Latest Developments and Future Directions

Cancer Epidemiology Biomarkers & Prevention, Journal Year: 2019, Volume and Issue: 28(7), P. 1095 - 1102

Published: April 30, 2019

Abstract Endometrial cancer, the most commonly diagnosed cancer of female reproductive tract in developed countries, has a heritable component. To date, 16 genetic risk regions have been robustly discovered by genome-wide association studies (GWAS) endometrial cancer. Post-GWAS analyses including expression quantitative trait loci analysis and laboratory-based functional successful identifying genes pathways involved carcinogenesis. Mendelian randomization confirmed factors causal for risk, increased body mass index early onset menarche. In this review, we summarize findings from GWAS post-GWAS We discuss clinical implications these findings, current knowledge gaps, future directions study genetics.

Language: Английский

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Comparisons of dual isogenic human iPSC pairs identify functional alterations directly caused by an epilepsy associated SCN1A mutation

Neurobiology of Disease, Journal Year: 2019, Volume and Issue: 134, P. 104627 - 104627

Published: Nov. 28, 2019

Over 1250 mutations in SCN1A, the Nav1.1 voltage-gated sodium channel gene, are associated with seizure disorders including GEFS+. To evaluate how a specific mutation, independent of genetic background, causes activity we generated two pairs isogenic human iPSC lines by CRISPR/Cas9 gene editing. One pair is control line from an unaffected sibling, and mutated carrying GEFS+ K1270T SCN1A mutation. The second patient corrected line. By comparing electrophysiological properties inhibitory excitatory iPSC-derived neurons these pairs, found mutation cell type-specific alterations current density evoked firing, resulting hyperactive neural networks. We also identified differences background interaction between background. Comparisons within dual neuronal cultures provide novel platform for evaluating cellular mechanisms underlying disease phenotype developing patient-specific anti-seizure therapies.

Language: Английский

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SOX9 haploinsufficiency reveals SOX9-Noggin interaction in BMP-SMAD signaling pathway in chondrogenesis

Cellular and Molecular Life Sciences, Journal Year: 2025, Volume and Issue: 82(1)

Published: March 2, 2025

Abstract Campomelic Dysplasia (CD) is a rare congenital disease caused by haploinsufficiency (HI) in SOX9. Patients with CD typically present skeletal abnormalities and 75% of them have sex reversal. In this study, we use CRISPR/Cas9 to generate human induced pluripotent stem cell (hiPSC) model from heathy male donor, based on previously reported SOX9 splice site mutation patients. This hiPSCs-derived chondrocytes heterozygotes (HT) homozygotes (HM) carriers showed significant defects chondrogenesis. Bulk RNA profiling revealed that the BMP-SMAD signaling pathway, ribosome-related, chromosome segregation-related gene sets were altered HT chondrocytes. The profile also noggin upregulation chondrocytes, ChIP-qPCR confirming binds distal regulatory element noggin. suggests plays feedback role BMP pathway modulating expression rather than acting solely as downstream regulator. provides further insights into its dosage sensitivity Overexpression promising results improved sulfated glycosaminoglycans (GAGs) aggregation COL2A1 following differentiation. We hope finding could provide better understanding dosage-dependent chondrogenesis contribute development therapeutic targets for

Language: Английский

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