Generation of two human ISG15 knockout iPSC clones using CRISPR/Cas9 editing

Merkert, Sylvia; Jaboreck, Mark-Christian; Engels, Lena; Malik, Muhammad Nasir Hayat; Göhring, Gudrun; Peßler, Frank; Martin, Ulrich; Olmer, Ruth

doi:10.1016/j.scr.2020.102135

Cited by 6 publications

(7 citation statements)

References 3 publications

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“…Considering that the antiviral effect of 4OI, BARD, and SFN is presumably mediated by activation of NRF2 signaling, we tested their impact on IAV infection in cells with a targeted inactivation of the NFE2L2 gene, which encodes NRF2 protein. We have previously shown that human induced pluripotent stem cells (hiPSC)-derived vascular endothelial cells (ECs) support IAV PR8 (H1N1) infection, but that viral transcription reaches lower levels than in A549 cells [ 16 ]. We inactivated the NFE2L2 gene in hiPSC by CRISPR/Cas9 editing and differentiated wild-type and NRF2 -/- cells into vascular ECs.…”

Section: Resultsmentioning

confidence: 99%

NRF2 activators inhibit influenza A virus replication by interfering with nucleo-cytoplasmic export of viral RNPs in an NRF2-independent manner

et al. 2023

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In addition to antioxidative and anti-inflammatory properties, activators of the cytoprotective nuclear factor erythroid-2-like-2 (NRF2) signaling pathway have antiviral effects, but the underlying antiviral mechanisms are incompletely understood. We evaluated the ability of the NRF2 activators 4-octyl itaconate (4OI), bardoxolone methyl (BARD), sulforaphane (SFN), and the inhibitor of exportin-1 (XPO1)-mediated nuclear export selinexor (SEL) to interfere with influenza virus A/Puerto Rico/8/1934 (H1N1) infection of human cells. All compounds reduced viral titers in supernatants from A549 cells and vascular endothelial cells in the order of efficacy SEL>4OI>BARD = SFN, which correlated with their ability to prevent nucleo-cytoplasmic export of viral nucleoprotein and the host cell protein p53. In contrast, intracellular levels of viral HA mRNA and nucleocapsid protein (NP) were unaffected. Knocking down mRNA encoding KEAP1 (the main inhibitor of NRF2) or inactivating the NFE2L2 gene (which encodes NRF2) revealed that physiologic NRF2 signaling restricts IAV replication. However, the antiviral effect of all compounds was NRF2-independent. Instead, XPO1 knock-down greatly reduced viral titers, and incubation of Calu3 cells with an alkynated 4OI probe demonstrated formation of a covalent complex with XPO1. Ligand–target modelling predicted covalent binding of all three NRF2 activators and SEL to the active site of XPO1 involving the critical Cys528. SEL and 4OI manifested the highest binding energies, whereby the 4-octyl tail of 4OI interacted extensively with the hydrophobic groove of XPO1, which binds nuclear export sequences on cargo proteins. Conversely, SEL as well as the three NRF2 activators were predicted to covalently bind the functionally critical Cys151 in KEAP1. Blocking XPO1-mediated nuclear export may, thus, constitute a “noncanonical” mechanism of anti-influenza activity of electrophilic NRF2 activators that can interact with similar cysteine environments at the active sites of XPO1 and KEAP1. Considering the importance of XPO1 function to a variety of pathogenic viruses, compounds that are optimized to inhibit both targets may constitute an important class of broadly active host-directed treatments that embody anti-inflammatory, cytoprotective, and antiviral properties.

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Section: Resultsmentioning

confidence: 99%

NRF2 activators inhibit influenza A virus replication by interfering with nucleo-cytoplasmic export of viral RNPs in an NRF2-independent manner

et al. 2023

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“…Wild‐type (WT) (MHHi001‐A) 27 and ISG15 –/– (MHHi001‐A‐3) hiPSCs 28 were maintained on mouse embryonic fibroblasts (CF1 MEF), which were used as feeder cells as previously described 29 or under feeder‐free conditions on Geltrex‐coated tissue culture flasks (TPP, Trasadingen, Switzerland) in E8 medium and passaged as single cells using Accutase™ (PAA, Pasching, Austria) with a seeding density of 3.6 × 10 4 cells/cm 2 . For differentiation toward macrophages embryoid body (EB) formation was induced.…”

Section: Methodsmentioning

confidence: 99%

ISG15 deficiency features a complex cellular phenotype that responds to treatment with itaconate and derivatives

Waqas

Sohail

Nguyen

et al. 2022

Clinical & Translational Med

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Background Congenital ISG15 deficiency is a rare autoinflammatory disorder that is driven by chronically elevated systemic interferon levels and predominantly affects central nervous system and skin. Methods and results We have developed induced pluripotent stem cell‐derived macrophages and endothelial cells as a model to study the cellular phenotype of ISG15 deficiency and identify novel treatments. ISG15 –/– macrophages exhibited the expected hyperinflammatory responses, but normal phagocytic function. In addition, they displayed a multifaceted pathological phenotype featuring increased apoptosis/pyroptosis, oxidative stress, glycolysis, and acylcarnitine levels, but decreased glutamine uptake, BCAT1 expression, branched chain amino acid catabolism, oxidative phosphorylation, β‐oxidation, and NAD(P)H‐dependent oxidoreductase activity. Furthermore, expression of genes involved in mitochondrial biogenesis and respiratory chain complexes II–V was diminished in ISG15 –/– cells. Defective mitochondrial respiration was restored by transduction with wild‐type ISG15, but only partially by a conjugation‐deficient variant, suggesting that some ISG15 functions in mitochondrial respiration require ISGylation to cellular targets. Treatment with itaconate, dimethyl‐itaconate, 4‐octyl‐itaconate, and the JAK1/2 inhibitor ruxolitinib ameliorated increased inflammation, propensity for cell death, and oxidative stress. Furthermore, the treatments greatly improved mitochondria‐related gene expression, BCAT1 levels, redox balance, and intracellular and extracellular ATP levels. However, efficacy differed among the compounds according to read‐out and cell type, suggesting that their effects on cellular targets are not identical. Indeed, only itaconates increased expression of anti‐oxidant genes NFE2L2, HMOX1 , and GPX7 , and dimethyl‐itaconate improved redox balance the most. Even though itaconate treatments normalized the elevated expression of interferon‐stimulated genes, ISG15 –/– macrophages maintained their reduced susceptibility to influenza virus infection. Conclusions These findings expand the cellular phenotype of human ISG15 deficiency and reveal the importance of ISG15 for regulating oxidative stress, branched chain amino acid metabolism, and mitochondrial function in humans. The results validate ruxolitinib as treatment for ISG15 deficiency and suggest itaconate‐based medications as additional therapeutics for this rare disorder.

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“…On the other hand, the use of this viral HDR template in HSPC mediates high toxicity through activation of the p53 pathway leading to impaired engraftment of the stem cells 51 . The most commonly used HDR template in iPSCs has been plasmid DNA, with sometimes striking negative effects on the editing e cacy [21][22][23] , which necessitate the subsequent use of selection strategies to enrich for gene-edited iPSC clones [22][23][24] . In our study we demonstrate that AAV6based HDR templates can be readily combined with the high activity of the Cas12a Ultra to correct disease-causing mutations in human iPSCs with high e ciency and without compromising the genome integrity.…”

Section: Discussionmentioning

confidence: 99%

“…However, HDR frequencies in iPSCs tended to be low, probably due to the use of plasmid-based HDR templates [21][22][23] . Several publications have highlighted the reliance on selection strategies to enrich for edited iPSC clones, which is labor-intensive, time-consuming, and may have a negative impact on the pluripotency and the genome integrity of the selected clones [22][23][24] . Furthermore, many protocols still involve the use of feeder cells in combination with antibiotics to enrich for corrected clones, which complicates the clinical translation of such cells and warrants the development of feeder-free and selection-free strategies.…”

Section: Introductionmentioning

confidence: 99%

Cas12a Ultra enables efficient genome editing in human multipotent and pluripotent stem cells

Hamad,

Alzubi,

Rhiel

et al. 2023

Preprint

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Background The development of the CRISPR-Cas12a platform has generated considerable interest in the genome editing community. Due to its AT-rich protospacer-adjacent motif (PAM, 5’-TTTV), Cas12a increased the potential number of targetable sites for gene editing beyond that of the prototypical Streptococcus pyogenes CRISPR-Cas9 system. Moreover, evaluation of the off-target activity of CRISPR-Cas12a nucleases suggested high specificity of the platform. Broad application of the CRISPR-Cas12a platform in primary human cells was recently enabled by the development of a re-engineered version of the natural Acidaminococcus Cas12a, called Cas12a Ultra. Methods We transferred the CRISPR-Cas12a Ultra system in the form of ribonucleoprotein complexes into clinically relevant human cells, including T cells, multipotent hematopoietic stem and progenitor cells (HSPCs), and induced pluripotent stem cells (iPSCs). Allelic gene editing frequencies were determined at various target sites using standard genotyping and next-generation sequencing. Furthermore, we evaluated targeted integration of transgenes into the AAVS1 safe harbor site and the CSF2RA locus of patient-derived iPSCs. Results We achieved allelic gene disruption frequencies of over 90% at various target sites in multiple primary human cell types. In addition, we demonstrated efficient knock-in of a GFP marker gene into the AAVS1 locus, and achieved targeted integration of a therapeutic DNA template into 90% of CSF2RA alleles in iPSCs without selection. Clonal analysis revealed bi-allelic integration in > 50% of the screened iPSC clones without compromising their pluripotency and genome integrity. Conclusions Herein, we demonstrate that the CRISPR-Cas12a Ultra system provides a highly efficient genome editing platform for human stem cell applications, expanding the toolbox for clinical applications.

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Generation of two human ISG15 knockout iPSC clones using CRISPR/Cas9 editing

Cited by 6 publications

References 3 publications

NRF2 activators inhibit influenza A virus replication by interfering with nucleo-cytoplasmic export of viral RNPs in an NRF2-independent manner

NRF2 activators inhibit influenza A virus replication by interfering with nucleo-cytoplasmic export of viral RNPs in an NRF2-independent manner

ISG15 deficiency features a complex cellular phenotype that responds to treatment with itaconate and derivatives

Cas12a Ultra enables efficient genome editing in human multipotent and pluripotent stem cells

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