Activation of the Anti-Aging and Cognition-Enhancing Gene Klotho by CRISPR-dCas9 Transcriptional Effector Complex

Abstract

Multiple lines of evidence show that the anti-aging and cognition-enhancing protein Klotho fosters neuronal survival, increases the anti-oxidative stress defense, and promotes remyelination of demyelinated axons. Thus, upregulation of the Klotho gene can potentially alleviate the symptoms and/or prevent the progression of age-associated neurodegenerative diseases such as Alzheimer’s disease and demyelinating diseases such as multiple sclerosis. Here we used a CRISPR-dCas9 complex to investigate single-guide RNA (sgRNA) targeting the Klotho promoter region for efficient transcriptional activation of the Klotho gene. We tested the sgRNAs within the − 1 to − 300 bp of the Klotho promoter region and identified two sgRNAs that can effectively enhance Klotho gene transcription. We examined the transcriptional activation of the Klotho gene using three different systems: a Firefly luciferase (FLuc) and NanoLuc luciferase (NLuc) coincidence reporter system, a NLuc knock-in in Klotho 3′-UTR using CRISPR genomic editing, and two human cell lines: neuronal SY5Y cells and kidney HK-2 cells that express Klotho endogenously. The two sgRNAs enhanced Klotho expression at both the gene and protein levels. Our results show the feasibility of gene therapy for targeting Klotho using CRISPR technology. Enhancing Klotho levels has a therapeutic potential for increasing cognition and treating age-associated neurodegenerative, demyelinating and other diseases, such as chronic kidney disease and cancer.

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Abbreviations

CRISPR:

clustered regularly interspaced short palindromic repeat

Cas:

CRISPR-associated

dCas9:

nuclease-deficient Cas9

sgRNA:

single-guide RNA

FLuc:

firefly luciferase

NLuc:

NanoLuc luciferase

3′-UTR:

3′-untranslated region

PCR:

polymerase chain reaction

DMEM:

Dulbecco’s modified Eagle’s Medium

PBS:

phosphate buffered saline

FBS:

fetal bovine serum

BSA:

bovine serum albumin

SDS-PAGE:

sodium dodecyl sulfate polyacrylamide gel electrophoresis

PGK:

phosphoglycerate kinase

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Acknowledgements

We thank Dr. Jason Nasse for reading and helpful suggestions for the manuscript.

Funding

This work was supported by NIH grants R56 AG051638, R44 AG053084 and R01 AG048927. 

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Correspondence to Carmela R. Abraham.

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Chen, CD., Zeldich, E., Li, Y. et al. Activation of the Anti-Aging and Cognition-Enhancing Gene Klotho by CRISPR-dCas9 Transcriptional Effector Complex. J Mol Neurosci 64, 175–184 (2018). https://doi.org/10.1007/s12031-017-1011-0

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Keywords

  • Alzheimer’s disease
  • Multiple sclerosis
  • Neuroprotection
  • Myelin
  • Chronic kidney disease
  • Cancer