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

Abstract

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.

Activation of pancreatic β-cell genes by multiplex epigenetic CRISPR-editing

“…In this study, we tested several parameters to determinate which are the best From all CRISPRa systems, dCas9-P300 was most effective at activating β -cell genes with reduced number of gRNAs. In concordance with previous work, we observed that dCas9-VP160 system requires several sgRNAs per gene to achieve a significant activation effect, 17,18,21 while dCas9-P300 reaches similar activation levels using only one sgRNA. 20 The different efficiencies could be attributed to the capacity of dCas9-P300 to induce a direct epigenetic modification, by acetylation of H3K27, while dCas-VP160 may result in an indirect effect by attracting the transcription machinery, which finally generates active epigenetic marks.…”

“…The algorithm used by this program is based on a previously described specificity analysis 34 . The INS sgRNAs and the PDX1 sgRNAs were previously reported 21,33 Plasmids and sgRNAs preparation All plasmid vectors used in this study were obtained from Addgene (plasmids #47108, #48226, #61357, #83889 and #82559 respectively). [18][19][20]35 The sgRNAs oligonucleotides containing the target sequences were cloned as described by Ran and colleagues.…”

“…Then, the DNA was treated with KruO4 for 5hmC recognition according to a previous report 38 and treated using a EpiTect Bisulfite Kit (Qiagen) as described. 21 The post-bisulfite promoter region of the human INS gene (NCBI ID 3630) was amplified by PCR using specific primers ( Supplementary Table S4).…”

“…VP64, VP160) 12,17,18 , similarly "epigenetic erasers" such as TET1 19 , and "epigenetic writers" such as the core of the p300 histone acetylation protein have been used in this system. 20 Our group and others found that using the CRISPR/dCas9 platform with VP160 effector and several sgRNA at the same time is associated with a synergistic stimulus for gene activation 17,18,21 . On the contrary, the use of CRISPR/dCas9 associated with p300 can be used efficiently with a single sgRNA.…”

CRISPR-targeted genome editing of mesenchymal stem cell-derived therapies for type 1 diabetes: a path to clinical success?

“…1). To this end, one study in particular has demonstrated success in activating endogenous human insulin transcription utilizing the dCas9-VP160 fusion and multiple insulin promoter targeting sgRNAs in HEK293T, Hela, and human fibroblasts [50]. The success of this study supports the proposed novel application of activating transcription of endogenous genes involved in pancreatic development such as Pdx-1 , Neurod1 , MafA , etc.…”