Analyzing CRISPR-Cas9 Genomic Engineering through Targeted Deletions – UROP Spring Symposium 2021

Analyzing CRISPR-Cas9 Genomic Engineering through Targeted Deletions

Ahsan Ahmed

Ahsan Ahmed

Pronouns: He/him

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 11
Presenter: 4

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Background: 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 CRISPR associated protein 9 (Cas9) nuclease that can cleave the targeted DNA complementary to a guide RNA (gRNA). Precise gene editing via CRISPR-Cas9 has great potential in treating inherited diseases (e.g., cystic fibrosis) or correcting genetic defects.

Objective: To test the specificity and function of the CRISPR-Cas9 in a proof-of-principle experiment, we used two human Emx1 (hEmx1) gene-specific gRNA sequences (3.1+4.1) to guide the Cas9 enzyme for the deletion of the targeted region in human 293AD cells.

Methodology: 293AD cells were first co-transfected with two plasmids that express the Cas9 and the hEmx1-targeting 3.1 or 4.1 gRNA. 72 hours after transfection, the DNA was extracted and purified from the cells. The targeted region of hEmx1 was amplified using a set of primers in the polymerase chain reaction (PCR). Cas-9 plus 3.1 gRNA plasmid alone transfection and GFP alone transfection were included as negative controls. The PCR products were run in a 0.8% agarose gel to check the presence of targeted deletion.

Results: Fluorescent microscopy showed efficient transfection of 293AD cells with the above plasmids. Agarose gel electrophoresis revealed two bands (639 bp for the WT product and 282 bp for the deleted product) in the Cas9 plus 3.1+4.1 lane. There was only one band of 639 bp for the other two control lanes.

Conclusions: The presence of the 282-bp deleted gene product demonstrates the efficiency of precise deletion of genomic DNA in mammalian cells using the CRISPR-Cas9 system.

Authors: Xin Tong, Ahsan Ahmed, Kyle Rodriguez
Research Method: Laboratory Research

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