CRISPR-Cas9 Based Saturation Mutagenesis Assay, Targeting Predicted Distal Transcriptional Control Elements Influencing the Cpe Gene

Michelle L Taylor

The importance of understanding genetic anomalies has become increasingly apparent, as knowledge of such complex concepts can lead to diagnosis and prevention of disease. The human Carboxypeptidase E gene (CPE) codes for a member of the M14 family of metallocarboxypeptidases and is 88% orthologous to the mouse Carboxypeptidase E gene (Cpe). Carboxypeptidase E has multiple functions, including the processing of proneurohormones and prohormones to produce bioactive peptides, in particular, insulin. Mechanisms such as DNA looping can bring distal promoters and enhancers physically close to genes, interact with, and illicit upregulation or maintenance of gene expression. It has been hypothesised that Carboxypeptidase E is under the influence of such distal control elements, and when such elements are compromised through CRISPR-Cas9 gene editing, gene expression levels of Carboxypeptidase E will become altered. This study aimed to employ a CRISPR-Cas9 based saturation mutagenesis assay to induce edits in predicted distal control regions, particularly enhancers, to determine if disruption of distal enhancers alters Carboxypeptidase E expression. Originally, sgRNAs were designed to target predicted pancreas/diabetes-related enhancers in the mouse mm10 genome, as Beta TC6 cells, insulin-producing mouse pancreas cells, were the proposed cell model for this study. However, due to issues outside of our control, the study was moved to look at the same designed sgRNAs in 3T3-L1 cells – mouse fibroblastic cells capable of chemically-induced differentiation to adipocytes. Upon inducing genome edits using CRISPR-Cas9 and these gRNAs in the fibroblastic 3T3-L1 cells, there was a statistically significant upregulation of Carboxypeptidase E expression (p = 2.2 x 10-16). This finding, although not what was originally expected, is nonetheless intriguing; discovering the identity/location of alternative distal control regions, in this case, repressors. Learning to manipulate distal control elements may prove useful in modulation of Carboxypeptidase E expression in its numerous roles, in modulation of Carboxypeptidase E’s role specifically in insulin processing, and potentially in the development of therapies for disease in which Carboxypeptidase E plays a role.

CRISPR-Cas9 Based Saturation Mutagenesis Assay, Targeting Predicted Distal Transcriptional Control Elements Influencing the Cpe Gene

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