Unexpectedly High Levels of Inverted Re-Insertions Using Paired sgRNAs for Genomic Deletions

Blayney, Joseph; Foster, Evangeline M.; Jagielowicz, Marta; Kreuzer, Mira; Morotti, Matteo; Reglinski, Katharina; Xiao, Julie; Hublitz, Philip

doi:10.3390/mps3030053

Cited by 12 publications

(8 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Mutations were verified by Sanger-sequencing of the subcloned amplicons, and absence of the respective protein was confirmed by immunoblotting. Of note, we obtained several clones in which the excised exon was found inverted and re-inserted, nevertheless giving a full KO phenotype as described (Blayney et al, 2020). All clones selected for this study were confirmed homozygous deletions.…”

Section: Methodsmentioning

confidence: 98%

Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5

Galiani

Reglinski

Carravilla

et al. 2021

Preprint

View full text Add to dashboard Cite

Measuring diffusion dynamics in living cells is essential for the understanding of molecular interactions. While various techniques have been used to explore such characteristics in the plasma membrane, this is less developed for measurements inside the cytosol. An example of cytosolic action is the import of proteins into peroxisomes, via the peroxisomal import receptor PEX5. Here, we combined advanced microscopy and spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and super-resolution STED microscopy to present a detailed characterization of the diffusion and interaction dynamics of PEX5. Among other features, we disclose a slow diffusion of PEX5, independent of aggregation or target binding, but associated with cytosolic interaction partners via its N-terminal domain. This sheds new light on the functionality of the receptor in the cytosol. Besides specific insights, our study highlights the potential of using complementary microscopy tools to decipher molecular interactions in the cytosol via studying their diffusion dynamics.

show abstract

Section: Methodsmentioning

confidence: 98%

Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5

Galiani

Reglinski

Carravilla

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…Cells were reprogrammed by exogenous expression of OCT3/4, SOX2, cMYC and KLF4 using a Sendai virus vector (41). To generate exon 2 -/-iPSCs (A4 and D1 clones), a dual single guide RNA (sgRNA) approach was chosen to recruit Cas9-2NLS (Synthego) to cut within intron 1 and 2 to remove CLU exon 2 (Additional le 1, table S1) (42). Transfection of CTR iPSCs was performed using Nucleofection and an optimised protocol (Synthego).…”

Section: Methodsmentioning

confidence: 99%

Glycosylated clusterin species facilitate Aβ toxicity in human neurons

Foster

Fernandes

Dangla-Valls

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

Background: Clusterin (CLU) is one of the most significant genetic risk factors for late onset Alzheimer’s disease. However, the mechanisms by which CLU contributes to AD development and pathogenesis remain unclear. Numerous studies have demonstrated that CLU-AD mutations and amyloid-β (Aβ) treatment alter the trafficking and localisation of glycosylated CLU proteins, which may contribute to AD pathogenesis. However, the roles of non-glycosylated and glycosylated CLU proteins in mediating Aβ toxicity have not been studied in human neurons. Methods: iPSCs with altered CLU trafficking were generated following the removal of CLU exon 2 by CRISPR/Cas9 gene editing. Neurons were generated from unedited wildtype, control unedited and exon 2 -/- edited iPSCs and were incubated with aggregated Aβ peptides. Changes in cell death and neurite length were quantified to determine if altered CLU protein trafficking influenced neuronal sensitivity to Aβ. Finally, RNA-Seq analysis was performed to identify key transcriptomic differences between CLU exon 2 -/- and CTR neurons.Results: The removal of CLU exon 2, and the ER-signal peptide located within, abolished the presence of glycosylated CLU and increased the abundance of intracellular, non-glycosylated CLU. Aβ25-35 treatment was demonstrated to alter glycosylated CLU trafficking in control neurons, while non-glycosylated CLU levels were unaltered in both, control and exon 2 -/- neurons. Partial protection against Aβ-induced cell death and neurite retraction was demonstrated in exon 2 -/- neurons. Transcriptome analysis identified downregulation of multiple extracellular matrix (ECM) related genes in exon 2 -/- neurons, potentially contributing to their reduced sensitivity to Aβ toxicity. Conclusions: This study identifies a crucial role of glycosylated CLU in facilitating Aβ toxicity in human neurons. The loss of these proteins reduced both, cell death and neurite damage, two key consequences of Aβ toxicity identified in the AD brain. Strikingly, transcriptomic differences between exon 2 -/- and CTR neurons were small, but a significant and consistent downregulation of ECM genes and pathways was identified in exon 2 -/- neurons. This may contribute to the reduced sensitivity of these neurons to Aβ, providing new mechanistic insights into Aβ pathologies and therapeutic targets for AD.

show abstract

“…The CTR iPSCs were previously generated from the Price lab at King's College London from human keratinocytes obtained from a healthy, neurotypical adult male, as previously described [45][46][47] . CTR iPSCs were used to generate exon 2 −/− iPSCs (A4 and D1 clones), a dual single guide RNA (sgRNA) approach was chosen to recruit Cas9-2NLS (Synthego) to cut within intron 1 and 2 to remove CLU exon 2 (Supplementary Table 1) 48 . Transfection of CTR iPSCs was performed using Nucleofection and an optimised protocol (Synthego).…”

Section: Methodsmentioning

confidence: 99%

Glycosylated clusterin species facilitate Aβ toxicity in human neurons

Foster

Fernandes

Dangla-Valls

et al. 2022

Sci Rep

Self Cite

View full text Add to dashboard Cite

Clusterin (CLU) is one of the most significant genetic risk factors for late onset Alzheimer’s disease (AD). However, the mechanisms by which CLU contributes to AD development and pathogenesis remain unclear. Studies have demonstrated that the trafficking and localisation of glycosylated CLU proteins is altered by CLU-AD mutations and amyloid-β (Aβ), which may contribute to AD pathogenesis. However, the roles of non-glycosylated and glycosylated CLU proteins in mediating Aβ toxicity have not been studied in human neurons. iPSCs with altered CLU trafficking were generated following the removal of CLU exon 2 by CRISPR/Cas9 gene editing. Neurons were generated from control (CTR) and exon 2 −/− edited iPSCs and were incubated with aggregated Aβ peptides. Aβ induced changes in cell death and neurite length were quantified to determine if altered CLU protein trafficking influenced neuronal sensitivity to Aβ. Finally, RNA-Seq analysis was performed to identify key transcriptomic differences between CLU exon 2 −/− and CTR neurons. The removal of CLU exon 2, and the endoplasmic reticulum (ER)-signal peptide located within, abolished the presence of glycosylated CLU and increased the abundance of intracellular, non-glycosylated CLU. While non-glycosylated CLU levels were unaltered by Aβ25–35 treatment, the trafficking of glycosylated CLU was altered in control but not exon 2 −/− neurons. The latter also displayed partial protection against Aβ-induced cell death and neurite retraction. Transcriptome analysis identified downregulation of multiple extracellular matrix (ECM) related genes in exon 2 −/− neurons, potentially contributing to their reduced sensitivity to Aβ toxicity. This study identifies a crucial role of glycosylated CLU in facilitating Aβ toxicity in human neurons. The loss of these proteins reduced both, cell death and neurite damage, two key consequences of Aβ toxicity identified in the AD brain. Strikingly, transcriptomic differences between exon 2 −/− and control neurons were small, but a significant and consistent downregulation of ECM genes and pathways was identified in exon 2 −/− neurons. This may contribute to the reduced sensitivity of these neurons to Aβ, providing new mechanistic insights into Aβ pathologies and therapeutic targets for AD.

show abstract

Unexpectedly High Levels of Inverted Re-Insertions Using Paired sgRNAs for Genomic Deletions

Cited by 12 publications

References 38 publications

Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5

Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5

Glycosylated clusterin species facilitate Aβ toxicity in human neurons

Glycosylated clusterin species facilitate Aβ toxicity in human neurons

Contact Info

Product

Resources

About