Generating Loss-of-function iPSC Lines with Combined CRISPR Indel  Formation and Reprogramming from Human Fibroblasts

Tidball, Andrew M.; Swaminathan, Preethi; Dang, Louis T.; Parent, Jack M.

doi:10.21769/bioprotoc.2794

Cited by 12 publications

(10 citation statements)

References 7 publications

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“…Manual picking of 50–100 colonies could take as much as an hour or more, especially for inexperienced users, while clonal isolation by FACS can be done in minutes. While manual passaging of hPSCs is not a common procedure anymore, manual picking of transfected clones still represents a common approach, especially for gene editing applications (Blair et al, 2016; Kwart et al, 2017; Wang et al, 2017; Howden et al, 2018; Tidball et al, 2018). By using this approach, single-cell sorting hPSCs by FACS following a transfection can be done with ease and clones may be expanded for genomic screening.…”

Section: Anticipated Resultsmentioning

confidence: 99%

An Efficient Protocol for Single-Cell Cloning Human Pluripotent Stem Cells

Singh

2019

Front. Cell Dev. Biol.

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Genomic manipulation of human pluripotent stem cells (hPSCs) has become essential to introduce genetic modifications and transgenes, and develop reporter lines. One of the major bottlenecks in gene editing is at the stage of single-cell cloning, which is thought to be variable across hPSC lines and is substantially reduced following a transfection. Due to the difficulty of performing fluorescent-assisted cell sorting (FACS) for single-cell isolation of hPSCs, previous approaches rely on manual colony picking, which is both time-consuming and labor-intensive. In this protocol, I provide a method for utilizing FACS to generate single-cell clones of hPSCs with efficiencies approaching 40% within 7–10 days. This can be achieved by sorting cells onto a feeder layer of MEFs in a stem cell defined medium with KSR and a Rock inhibitor, as early as 1–2 days following a transfection, streamlining the gene editing process. The approach described here provides a fundamental method for all researchers utilizing hPSCs for scientific studies.

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Section: Anticipated Resultsmentioning

confidence: 99%

An Efficient Protocol for Single-Cell Cloning Human Pluripotent Stem Cells

Singh

2019

Front. Cell Dev. Biol.

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“…We used CRISPR gene editing ( Tidball et al., 2017 , Tidball et al., 2018 ) to generate a heterozygous deletion in SCN1A ( SCN1A + / − ) in a control line to ask whether Nav1.1 haploinsufficiency alone was sufficient to increase I Na in iPSC-CMs, or whether this result was dependent on genetic background. The mutant clone used in this study had a heterozygous deletion of a cytosine at position 23 of the SCN1A cDNA (c.23del in NM_001165963.2), resulting in a mutation with a proline changed to a histidine at position 8, and a frameshift resulting in a premature stop codon at position 91 (p.P8HfsTer91) ( Figure S4 ).…”

Section: Resultsmentioning

confidence: 99%

Channelopathy as a SUDEP Biomarker in Dravet Syndrome Patient-Derived Cardiac Myocytes

et al. 2018

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SummaryDravet syndrome (DS) is a severe developmental and epileptic encephalopathy with a high incidence of sudden unexpected death in epilepsy (SUDEP). Most DS patients carry de novo variants in SCN1A, resulting in Nav1.1 haploinsufficiency. Because SCN1A is expressed in heart and in brain, we proposed that cardiac arrhythmia contributes to SUDEP in DS. We generated DS patient and control induced pluripotent stem cell-derived cardiac myocytes (iPSC-CMs). We observed increased sodium current (INa) and spontaneous contraction rates in DS patient iPSC-CMs versus controls. For the subject with the largest increase in INa, cardiac abnormalities were revealed upon clinical evaluation. Generation of a CRISPR gene-edited heterozygous SCN1A deletion in control iPSCs increased INa density in iPSC-CMs similar to that seen in patient cells. Thus, the high risk of SUDEP in DS may result from a predisposition to cardiac arrhythmias in addition to seizures, reflecting expression of SCN1A in heart and brain.

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“…We have previously published methods combining CRISPR genome-editing and iPSC reprogramming (Tidball et al, 2017;Tidball et al, 2018). See Supplemental materials for further details describing the generation of the P2 rescue iPSC line.…”

Section: Crispr Genome-editing With Simultaneous Reprogrammingmentioning

confidence: 99%

Variant-specific changes in persistent or resurgent Na⁺current inSCN8A-EIEE13 iPSC-derived neurons

Tidball

Yuan

et al. 2020

Preprint

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Missense variants in the voltage-gated sodium channel (VGSC) gene, SCN8A, are linked to earlyinfantile epileptic encephalopathy type 13 (EIEE13). EIEE13 patients exhibit a wide spectrum of intractable seizure types, severe developmental delay, movement disorders, and elevated risk of sudden unexpected death in epilepsy (SUDEP). The mechanisms by which SCN8A variants lead to epilepsy are poorly understood, although heterologous expression systems and mouse models have demonstrated altered sodium current (INa) properties. To investigate these mechanisms using a patient-specific model system, we generated induced pluripotent stem cells (iPSCs) from three patients with missense variants in SCN8A: p.R1872>L (P1); p.V1592>L (P2); and p.N1759>S (P3). Using small molecule differentiation into excitatory neurons, iPSC-derived neurons from all three patients displayed altered INa. P1 and P2 had elevated persistent INa, while P3 had increased resurgent INa compared to controls. Further analyses focused on one of the patients with increased persistent INa (P1) and the patient with increased resurgent INa (P3). Excitatory cortical neurons from both patients had prolonged action potential (AP) repolarization and shorter axon initial segment lengths compared to controls, the latter analyzed by immunostaining for ankyrin-G. Using doxycycline-inducible expression of the neuronal transcription factors Neurogenin 1 and 2 to synchronize differentiation of induced excitatory cortical-like neurons (iNeurons), we investigated network activity and response to pharmacotherapies. Both patient neurons and iNeurons displayed similar abnormalities in AP repolarization. Patient iNeurons showed increased burstiness that was sensitive to phenytoin, currently a standard treatment for EIEE patients, or riluzole, an FDAapproved drug used in amyotrophic lateral sclerosis and known to block persistent and resurgentINa, at pharmacologically relevant concentrations. Patch-clamp recordings showed that riluzole suppressed spontaneous firing and increased the AP firing threshold of patient-derived neurons to more depolarized potentials. Our results indicate that patient-specific neurons are useful for modeling EIEE13 and demonstrate SCN8A variant-specific mechanisms. Moreover, these findings suggest that patient-specific iPSC neuronal disease modeling offers a useful platform for discovering precision epilepsy therapies. Table 1. Patient information. Information for patients and controls from which the iPSC lines were derived are presented including, age at biopsy, sex, SCN8A de novo variant, seizure type, medications, and other findings.

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Generating Loss-of-function iPSC Lines with Combined CRISPR Indel Formation and Reprogramming from Human Fibroblasts

Cited by 12 publications

References 7 publications

An Efficient Protocol for Single-Cell Cloning Human Pluripotent Stem Cells

An Efficient Protocol for Single-Cell Cloning Human Pluripotent Stem Cells

Channelopathy as a SUDEP Biomarker in Dravet Syndrome Patient-Derived Cardiac Myocytes

Variant-specific changes in persistent or resurgent Na⁺current inSCN8A-EIEE13 iPSC-derived neurons

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Generating Loss-of-function iPSC Lines with Combined CRISPR Indel Formation and Reprogramming from Human Fibroblasts

Cited by 12 publications

References 7 publications

An Efficient Protocol for Single-Cell Cloning Human Pluripotent Stem Cells

An Efficient Protocol for Single-Cell Cloning Human Pluripotent Stem Cells

Channelopathy as a SUDEP Biomarker in Dravet Syndrome Patient-Derived Cardiac Myocytes

Variant-specific changes in persistent or resurgent Na+current inSCN8A-EIEE13 iPSC-derived neurons

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Variant-specific changes in persistent or resurgent Na⁺current inSCN8A-EIEE13 iPSC-derived neurons