RESUMO
BACKGROUND: Prime editing (PE) is the most recent gene editing technology able to introduce targeted alterations to the genome, including single base pair changes, small insertions, and deletions. Several improvements to the PE machinery have been made in the past few years, and these have been tested in a range of model systems including immortalized cell lines, stem cells, and animal models. While double nicking RNA (dncRNA) PE systems PE3 and PE5 currently show the highest editing rates, they come with reduced accuracy as undesired indels or SNVs arise at edited loci. Here, we aimed to improve single ncRNA (sncRNA) systems PE2 and PE4max by generating novel all-in-one (pAIO) plasmids driven by an EF-1α promoter, which is especially suitable for human-induced pluripotent stem cell (hiPSC) models. RESULTS: pAIO-EF1α-PE2 and pAIO-EF1α-PE4max were used to edit the voltage gated potassium channel gene KCNQ2 and voltage gated sodium channel gene SCN1A. Two clinically relevant mutations were corrected using pAIO-EF1α-PE2 including the homozygous truncating SCN1A R612* variant in HEK293T cells and the heterozygous gain-of-function KCNQ2 R201C variant in patient-derived hiPSC. We show that sncRNA PE yielded detectable editing rates in hiPSC ranging between 6.4% and 9.8%, which was further increased to 41% after a GFP-based fluorescence-activated cell sorting (FACS) cell sorting step. Furthermore, we show that selecting the high GFP expressing population improved editing efficiencies up to 3.2-fold compared to the low GFP expressing population, demonstrating that not only delivery but also the number of copies of the PE enzyme and/or pegRNA per cell are important for efficient editing. Edit rates were not improved when an additional silent protospacer-adjacent motif (PAM)-removing alteration was introduced in hiPSC at the target locus. Finally, there were no genome-wide off-target effects using pAIO-EF1α-PE2 and no off-target editing activity near the edit locus highlighting the accuracy of snc prime editors. CONCLUSION: Taken together, our study shows an improved efficacy of EF-1α driven sncRNA pAIO-PE plasmids in hiPSC reaching high editing rates, especially after FACS sorting. Optimizing these sncRNA PE systems is of high value when considering future therapeutic in vivo use, where accuracy will be extremely important.
Assuntos
Sistemas CRISPR-Cas , Pequeno RNA não Traduzido , Animais , Humanos , Células HEK293 , Fator 1 de Elongação de Peptídeos/genética , Plasmídeos/genética , Canal de Potássio KCNQ2/genética , Canal de Sódio Disparado por Voltagem NAV1.1/genéticaRESUMO
There are currently seven different zebrafish strains that model Dravet Syndrome, a severe childhood form of epilepsy. These models are based on a set of duplicated genes, scn1laa and scn1lab, which are the homologs for human SCN1A. Disrupting one of the genes would mimic a heterozygous disease state in humans, as the paralog gene is still present. While this 'disease-state model' is widely accepted, there is also evidence that the function of these genes might not be completely the same. By analyzing the functional domains, we discovered several hotspots in the protein that are not conserved, indicating a functional difference. Based on this, we generated scn1Laa knockout zebrafish and compared their phenotype to scn1lab knockouts. The genetic and functional differences we discovered can have implications for the use of zebrafish as a model for Dravet Syndrome.
Assuntos
Epilepsias Mioclônicas , Canal de Sódio Disparado por Voltagem NAV1.1 , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Modelos Animais de Doenças , Epilepsias Mioclônicas/genética , Humanos , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Dravet syndrome (DS) is a monogenic epileptic encephalopathy caused by loss-of-function mutations in the voltage-gated sodium channel (VGSC) gene SCN1A. DS has an age of onset within the first year of life and severe disease prognosis. In the past years, it has been shown that upregulation of endogenous SCN1A can be beneficial in animal models for DS, but a complete rescue was not observed. We hypothesized that upregulation during early development that precedes onset of first symptoms might improve disease outcome. To test this hypothesis, we first evaluated the CRISPR activating method for early upregulation of voltage gated sodium channels during early development. We injected CRISPRa components, which target the proximal or distal promoter region of the VGSC gene scn1Laa in the yolk of one-cell stage zebrafish embryos. The effect of both dCas9-VPR and dCas9-VP64 was evaluated. Both CRISPRa fusions showed toxicity in the majority of embryos, with or without guide RNAs. The few embryos that survived developed normally, and dCas9-VPR induces an upregulation of scn1Laa mRNA until 24 hours after fertilization. At 5 days post fertilization, CRISPRa-injected embryos showed an epileptic phenotype, including locomotor burst movements, hyperactivity, and epileptiform activity originating from the brain. In addition to previously published scn1Laa and scn1Lab loss-of-function models, we conclude that gain of scn1Laa function can have an equally severe phenotype. Upregulation of scn1Laa in the current zebrafish model for DS, scn1Lab-KO, aggravated the disease phenotype, highlighting that early-stage upregulation using CRISPRa can lead to both toxicity and a worsening of the disease phenotype.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Desenvolvimento Embrionário/genética , Epilepsia/etiologia , Regulação da Expressão Gênica , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Fenótipo , Proteínas de Peixe-Zebra/genética , Animais , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Suscetibilidade a Doenças , Epilepsias Mioclônicas/diagnóstico , Epilepsias Mioclônicas/etiologia , Epilepsia/diagnóstico , Edição de Genes , Estudos de Associação Genética , RNA Guia de Cinetoplastídeos , RNA Mensageiro , Peixe-ZebraRESUMO
Dravet syndrome is caused by dominant loss-of-function mutations in SCN1A which cause reduced activity of Nav1.1 leading to lack of neuronal inhibition. On the other hand, gain-of-function mutations in SCN8A can lead to a severe epileptic encephalopathy subtype by over activating NaV1.6 channels. These observations suggest that Nav1.1 and Nav1.6 represent two opposing sides of the neuronal balance between inhibition and activation. Here, we hypothesize that Dravet syndrome may be treated by either enhancing Nav1.1 or reducing Nav1.6 activity. To test this hypothesis we generated and characterized a novel DS zebrafish model and tested new compounds that selectively activate or inhibit the human NaV1.1 or NaV1.6 channel respectively. We used CRISPR/Cas9 to generate two separate Scn1Lab knockout lines as an alternative to previous zebrafish models generated by random mutagenesis or morpholino oligomers. Using an optimized locomotor assay, spontaneous burst movements were detected that were unique to Scn1Lab knockouts and disappear when introducing human SCN1A mRNA. Besides the behavioral phenotype, Scn1Lab knockouts show sudden, electrical discharges in the brain that indicate epileptic seizures in zebrafish. Scn1Lab knockouts showed increased sensitivity to the GABA antagonist pentylenetetrazole and a reduction in whole organism GABA levels. Drug screenings further validated a Dravet syndrome phenotype. We tested the NaV1.1 activator AA43279 and two novel NaV1.6 inhibitors MV1369 and MV1312 in the Scn1Lab knockouts. Both type of compounds significantly reduced the number of spontaneous burst movements and seizure activity. Our results show that selective inhibition of NaV1.6 could be just as efficient as selective activation of NaV1.1 and these approaches could prove to be novel potential treatment strategies for Dravet syndrome and other (genetic) epilepsies. Compounds tested in zebrafish however, should always be further validated in other model systems for efficacy in mammals and to screen for potential side effects.