Abstract
After damage to the adult mammalian central nervous system (CNS), surviving neurons have limited capacity to regenerate and restore functional connectivity. Conditional genetic deletion of PTEN results in robust CNS axon regrowth, while PTEN repression with short hairpin RNA (shRNA) improves regeneration but to a lesser extent, likely due to suboptimal PTEN mRNA knockdown using this approach. Here we employed the CRISPR/dCas9 system to repress PTEN transcription in neural cells. We targeted the PTEN proximal promoter and 5âČ untranslated region with dCas9 fused to the repressor protein KrĂŒppel-associated box (KRAB). dCas9-KRAB delivered in a lentiviral vector with one CRISPR guide RNA (gRNA) 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. 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, and enhanced neurite outgrowth after nerve growth factor stimulation. PTEN silencing with CRISPR/dCas9 epigenetic editing may provide a new option for promoting axon regeneration and functional recovery after CNS trauma.
Highlights
The devastating consequences of physical trauma, stroke or chronic neurodegenerative disease on central nervous system (CNS) function are largely due to the lack of effective repair mechanisms and the inability to regenerate neural circuitry after damage
We investigated PTEN repression in human cell line models, neural stem cells and in induced pluripotent stem cell-derived CNS neurons using a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) epigenetic editing system
We designed four guide RNA (gRNA) targeting the Homo sapiens PTEN proximal promoter and 5âČ untranslated region (UTR), two of which we had previously used for transcriptional activation of PTEN49 (Fig. 1C, Supplementary Table S1). gRNA target sites were selected for minimal predicted off-target activity and maximal on-target activity according to established Âalgorithms[50]
Summary
The devastating consequences of physical trauma, stroke or chronic neurodegenerative disease on central nervous system (CNS) function are largely due to the lack of effective repair mechanisms and the inability to regenerate neural circuitry after damage. ShRNAs targeting PTEN have been delivered to the injured spinal cord or optic nerve by adeno-associated virus (AAV), resulting in some regeneration of damaged axons which formed synapses in target regions distal to the injury Âsite[25,26]. In these studies PTEN shRNA showed only modest levels of knockdown of PTEN, and the extent of axon regeneration was not as significant as with genetic PTEN deletion, likely due to residual PTEN Âexpression[25,26]. The CRISPR system has been adapted for transcriptional activation, repression, and epigenetic editing by mutations to the catalytic domains of Cas[9] to form a âdeadâ Cas[9] (dCas9) protein, which binds the DNA target specified by the gRNA without initiating a double-strand break. KRAB recruits KRAB-associated protein 1 (KAP1), thereby engaging histone deacetylases (HDACs) and histone methyltransferases (HMTs) to promote heterochromatin formation (Fig. 1B)36â41. dCas9-KRAB fusion proteins reduce H3K9 and H3K27 acetylation, increase H3K9 and H3K27 trimethylation, and reduce chromatin accessibility at Scientific Reports | (2020) 10:11393 |
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