Generation and validation of lentiviral CRISPR library for E3 ubiquitin ligases

Abstract

Transcription Factor 4 (TCF4) is a protein belonging to the bHLH family widely expressed in several tissues during development. The function of TCF4 in the nervous system is related to the proliferation and differentiation of neural progenitor cells, and for that reason, mutations in the TCF4 gene are associated with countless neurological disorders. The haploinsufficiency of the Transcription Factor 4 causes Pitt-Hopkins Syndrome (PTHS), a rare genetic neurological disorder belonging to the autistic spectrum witch manifestations include repetitive and stereotyped behaviors, motor delay, cognitive deficit, gastrointestinal anomalies, and a distinctive facial phenotype. Although PTHS impact the patients' lives and their families, there isn't still a specific treatment. Since the PTHS is caused by the insufficient amount of the TCF4 protein in the cells, the understanding of the TCF4 degradation becomes extremely relevant for new therapies development. The ubiquitination process is a pos-translation modification that occurs through an enzymatic reaction chain involving E1, E2, and E3 enzymes, which leads to protein labeling for the degradation via the proteasome. The E3 ubiquitin ligases perform the last step in the process, promoting specificity to the reaction by recognizing the lysine residues in target proteins and catalyzing the ubiquitin molecule transference. To find the E3 ubiquitin ligase involved in the TCF4 degradation process, the laboratory will perform a screening of the CRISPR library in lentiviral vectors including sgRNAs for all the E3 ubiquitin ligases of the human genome in neural progenitor cells (NPCs) overexpression the TCF4 protein fused to a gene reporter. CRISPR screening is a technique based on large-scale genetic editions for the generation of the knockout in selected target genes, which aim is the obtaining a heterogeneous cell population, where each cell contains knockout for a different gene. The current Scientific Initiation project has as its objective to produce the lentiviruses of the CRISPR library for E3 ubiquitin ligases and validate these vectors that will be in the future used in studies that seek to identify the enzyme involved in the TCF4 degradation process via the proteasome.