Modulation of Insulin Gene Expression with CRISPR/Cas9-based Transcription Factors

Abstract

Background: The discovery and use of CRISPR/Cas9 technology have enabled researchers throughout the globe to continuously edit genomes for the benefit of science and medicine. Diabetes type I is one field of medicine where CRISPR/Cas9 has a strong potential for cell therapy development. The long-lasting paucity of healthy cells for clinical transplantation into diabetic patients has led to the search of new methods for producing β-cells from other human cell types. Embryonic stem cells are being studied worldwide as one most promising solution of this need. Aim: The aim of the study is to to check the feasibility of modulating human insulin transcription using CRISPR/Cas9-based synthetic transcription regulation factors.
 Results: A new approach for creating potential therapeutic donor cells with enhanced and suppressed insulin production based on one of the latest achievements of human genome editing was developed. Both synthetic transcription activator (VP64) and transcription repressor (KRAB) proteins were shown to function adequately well as a part of the whole CRISPR/Cas9-based system. We claim that our results have a lot to offer and can bring light to many studies where numerous labs are struggling on to treat this disease.

Latest advancements in the development of new therapies for type 1 diabetes

“…Interleukin 10, an anti-inflammatory cytokine, has been shown to reduce autoimmune damage to β-cells and enhance the resilience of stem cells as part of the immune system of recipients after transplantation. Additionally, in 2021, Alzhanuly et al demonstrated the application of CRISPR-Cas9 technology in human cells to modulate insulin production (43).…”

Challenges of CRISPR/Cas-Based Cell Therapy for Type 1 Diabetes: How Not to Engineer a “Trojan Horse”

“…The most active among these factors were found to be pancreatic and duodenal homeobox 1 (PDx1), protoendocrine factor (neurogenin 3, Ngn3), NK6 homeobox 1 (NKx6.1), and musculoaponeurotic fibrosarcoma oncogene family A (MaFA). In further experiments, the possibility of manipulating insulin production in HEK293 cells was demonstrated through the artificial activation of the INS insulin gene with CRISPR/dCas9-based transcription factors [249]. The stimulation of the insulin production has been achieved in pancreatic β-cell genes through the multiplex activation of PDX1, NEUROG3, PAX4, and INS genes using CRISPR/dCas9-VP160, CRISPR/dCas9-TET1 and CRISPR/dCas9-P300 epigenetic activators [250].…”

State-of-the-art in engineering small molecule biosensors and their applications in metabolic engineering

“…Metabolic controllers based on induced protein degradation have the potential to overcome issues related to the accumulation of inhibitory or toxic intermediates and the heterogeneity of cellular states and environments by preventing accumulation of causal enzymes through negative feedback control. Protein degradation systems offer the advantage of transcriptional independence over other exogenous approaches for modulating protein activity, such as transcriptional repression [138], RNAi [139,140], CRISPR/Cas9 and/or dCas9 [141,142], toggle switches [139,143]. Knocking out, silencing, or turning off protein-encoding genes often disrupts cognate genes, leading to slow growth phenotypes and deleterious effects on cell metabolism [135].…”