O uso da ferramenta CRISPR/dCas9 para reprimir e superexpressar progranulina em células humanas

Abstract

Schizophrenia is a severe psychiatric disorder that affects about 23.6 million people worldwide, with an estimated worldwide prevalence of 0.3% to 0.7%. People with this pathology may present positive symptoms, such as hallucinations and delusions, negative symptoms, including depressive behaviors, disordered thinking, and speech and social withdrawal, as well as cognitive deficits, such as attention and learning problems. In addition, schizophrenia is considered to be a multifactorial disorder, with genetic factors and environmental stressors acting together, making individuals prone to develop this disease. Previous published studies suggest that the GRN gene, which encodes a glycoprotein known as Progranulin, may be associated with an increased risk of developing schizophrenia. Previous unpublished data from our research group shows that induced microglia-like cells (iMGs) derived from individuals with schizophrenia express lower levels of Progranulin compared to those derived from healthy individuals. Thus, this study aims at establishing a functional platform using the CRISPR/dCas9 system to modulate the transcription of this gene. To accomplish that, we employed both CRISPRa and CRISPRi systems, using the dCas9 protein fused with the transcriptional effectors VPR and KRAB, respectively. The present data demonstrates the proper functioning of the CRISPR/dCas9 system, making it possible to transcriptionally modulate the GRN gene in central nervous system cells. Interestingly, we observed that astrocytes derived from individuals with schizophrenia, as well as iMGs, also exhibit dysregulation in Progranulin expression levels. Astrocytes derived from individuals with schizophrenia produced higher levels of Progranulin mRNA when stimulated with TNF-α compared to astrocytes from healthy individuals. Therefore, we can use the transcriptional regulation tools developed in this study to modulate Progranulin expression in cells derived from individuals with schizophrenia, aiming to better understand the importance of this protein in schizophrenia.