Research Mentor(s): Xin Tong, Research Assistant Professor
Research Mentor School/College/Department: Molecular & Integrative Physiology, Michigan Medicine
Presentation Date: Thursday, April 22, 2021
Session: Session 6 (4pm-4:50pm)
Breakout Room: Room 7
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a technology used to edit genomes at very high precision. It enables precise editing of genomic loci with a RNA-guided Cas9 nuclease that can cleave target DNA that is complementary to a guide RNA (gRNA). Then, the CRISPR-Cas9 system can be used to achieve various goals such as treating inherited diseases (e.g., cystic fibrosis). The objective is to test the specificity and function of the CRISPR-Cas9 system in deleting a targeted sequence in mammalian cells. Mammalian 293AD cells were first transfected with the Cas9-expressing plasmid and human EMX1 ()-specific 3.1+4.1 gRNA sequences. 72 hours after transfection, the DNA was extracted and purified from the cells. The hEMX1 modified region was amplified using a set of primers in the polymerase chain reaction (PCR). The PCR products were run in a 2% agarose gel for gel electrophoresis where the sizes of the DNA fragments were compared to a marker. Results from gel electrophoresis showed that the Cas9 system was successfully able to target the hEMX1 gene when 3.1+4.1 gRNA was present. However, pNTAP posi-tag, a plasmid without the hEMX1 sequence, also showed unexpected cuts which lead to DNA fragments of various sizes. The results show that the CRISPR-Cas9 system was propitious in targeting the hEMX1 DNA sequence. The unexpected cuts made in pNTAP posi-tag will require further experimentation, with one possibility being to extract, purify, and sequence the unanticipated DNA fragments in order to explain the results.