Transcriptional repression of PTEN in neural cells using CRISPR/dCas9 epigenetic editing

Scientific Reports, 2020 · DOI: 10.1038/s41598-020-68257-y · Published: July 15, 2020

Simple Explanation

The study uses CRISPR/dCas9 system to repress PTEN transcription in neural cells. They targeted the PTEN proximal promoter and 5′ untranslated region with dCas9 fused to the repressor protein Krüppel-associated box (KRAB). The dCas9-KRAB system outperformed a combination of four shRNAs targeting the PTEN transcript, a construct previously used in CNS injury models. The CRISPR system also worked more effectively than shRNAs for Pten repression in rat neural crest-derived PC-12 cells PTEN silencing with CRISPR/dCas9 epigenetic editing may provide a new option for promoting axon regeneration and functional recovery after CNS trauma.

Study Duration

Not specified

Participants

Human cell line models, neural stem cells and in induced pluripotent stem cell (iPSC)-derived CNS neurons, rat neural crest-derived PC-12 cells

Evidence Level

Not specified

Key Findings

1
dCas9-KRAB achieved potent and specific PTEN repression in human cell line models and neural cells derived from human iPSCs, and induced histone (H)3 methylation and deacetylation at the PTEN promoter.
2
The dCas9-KRAB system outperformed a combination of four shRNAs targeting the PTEN transcript.
3
The CRISPR system also worked more effectively than shRNAs for Pten repression in rat neural crest-derived PC-12 cells, and enhanced neurite outgrowth after nerve growth factor stimulation.

Research Summary

The study demonstrates that dCas9-KRAB, targeted to the PTEN 5′ UTR by a single gRNA, potently and specifically repressed PTEN expression at mRNA and protein level. The dCas9-KRAB system repressed PTEN to a greater extent than a combination of four PTEN-targeting shRNAs in several experimental human cell types. Delivery of the CRISPR system to hNSCs or hNSC-derived neurons successfully repressed PTEN expression without altering expression of NSC-specific or neuron-specific markers.

Practical Implications

Potential Therapeutic Strategy

PTEN silencing with CRISPR/dCas9 epigenetic editing may provide a new option for promoting axon regeneration and functional recovery after CNS trauma.

Superiority over shRNA

The CRISPR/dCas9 system achieved extremely potent repression in human cells, and could provide a strategy for PTEN inhibition that is almost as effective as PTEN genetic deletion, but with far greater translational potential, due to its reversibility and the reduced risk of exogenous DNA integration into Cas9-induced double-strand breaks.

Specific Gene Regulation

The gRNAs employed here did not affect expression of predicted off-target genes, and many other studies support the claim that transcriptional regulation with dCas9 is highly specific.

Study Limitations

1
Cultured neurons do not provide definitive information as to whether dCas9-KRAB can promote axon regeneration of damaged CNS neurons in vivo
2
PTEN repression may not be safe or desirable beyond the point of axon regeneration and the reformation of connections with target neurons.
3
There is the possibility that constitutive and permanent PTEN knockdown could lead to cancer development, as PTEN has well-established tumor suppressive functions

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