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CRISPR-on system for the activation of the endogenous human INS gene


Advances in the field of epigenetics have allowed the design of new therapeutic strategies to address complex diseases such as type 1 diabetes (T1D). Clustered regularly interspaced short palindromic repeats (CRISPR)-on is a novel and powerful RNA-guided transcriptional activator system that can turn on specific gene expression; however, it remains unclear whether this system can be widely used or whether its use will be restricted depending on cell types, methylation promoter statuses or the capacity to modulate chromatin state. Our results revealed that the CRISPR-on system fused with transcriptional activators (dCas9-VP160) activated endogenous human INS, which is a silenced gene with a fully methylated promoter. Similarly, we observed a synergistic effect on gene activation when multiple single guide RNAs were used, and the transcriptional activation was maintained until day 21. Regarding the epigenetic profile, the targeted promoter gene did not exhibit alteration in its methylation status but rather exhibited altered levels of H3K9ac following treatment. Importantly, we showed that dCas9-VP160 acts on patients’ cells in vitro, particularly the fibroblasts of patients with T1D.

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This work is dedicated to the memory of Dr Pablo Argibay. CG and FPB were financed by CONICET. We thank Dr Masaru Okabe, Osaka University, for kindly providing the pCX-EGFP plasmid. We thank Nelson Argumedo Rueda, Hospital Italiano, for help in the preparation of this manuscript.

Author contributions

CG, PA and FPB conceived and designed the experiments. CG, MI and FPB performed the experiments. CG and FPB analyzed the data. AM and LG contributed reagents/materials/analysis tools. CG and FPB wrote the paper. FPB is the guarantor of this work and as such had full access to all of the data in the study and assumes responsibility for the integrity of the data and the accuracy of the data analysis.

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Correspondence to F Pereyra-Bonnet.

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The authors declare no conflict of interest.

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Supplementary Information accompanies this paper on Gene Therapy website

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Giménez, C., Ielpi, M., Mutto, A. et al. CRISPR-on system for the activation of the endogenous human INS gene. Gene Ther 23, 543–547 (2016).

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