Utilization of CRISPR-CAS as a Genome Editing Tool to Encode the Notch1-mNeonGreen Fusion Protein in Mammalian Cells

Clustered regularly interspaced palindromic repeats (CRISPR-Cas) is an example of DNA editing technology that is naturally found in the immune system of bacteria but has been engineered to edit DNA sequences in mammalian cells. This can be used by medical scientists to treat genetic diseases like sickle cell anemia. The popularity of using CRISPR-Cas has increased due to its ability to add/delete any sequence that is located in a specific site in the genome, called the target sequence. The target sequence chosen for this project was the Notch1 gene that encodes the Notch1 protein, which is part of the Notch signaling pathway vital for the embryonic development of tissues throughout the body. In this project, CRISPR-Cas will be used to generate a new line of mammalian cells by inserting a fluorescent protein sequence (mNeonGreen) into the Notch1 sequence, hopefully creating cells with a Notch1-mNeonGreen fused protein.

Three target sequences were designed to target the Notch1 sequence in the mammalian genome. The target sequences were combined with circular pieces of DNA (plasmids) that contain the Cas9 enzyme to generate a complex that will target and cut the double strands of DNA that encodes the Notch1 protein to insert the mNeonGreen sequence. For future work, the complex with the target sequence and Cas9 enzyme needs to be combined with a plasmid that contains the mNeonGreen sequence, so it can be taken up by mammalian cells, potentially generating a new line of cells that express the Notch1-mNeonGreen fused protein.