In Utero Gene Editing: CRISPR-Cas9 and MTMG

Students

2019
College

Faculty

Assistant Professor of Surgery

Project Summary

PURM has given me the wonderful opportunity to work on the latest, most innovative research. Dr. William Peranteau’s lab focuses on in utero research, and my project specifically revolved around the efficacy of gene editing when performed in utero.

CRISPR-Cas9 is a gene editing tool regarded to be highly accurate and able to repair, insert, or delete genes within the genome. Although several studies have already shown its potential in editing genes in vivo and in vitro, its potential in an in utero setting is not as clear.

This summer, I assisted with the lab’s MTMG mouse project. The MTMG mouse model is a special type of mouse that contains two genes, “mT” (membrane-targeted tomato) and “mG” (membrane targeted green), which lie in tandem within a chromosome. When both genes are present, the mice organs will fluoresce red. When the mT gene is removed, however, the mice organs fluoresce green, giving us a visual representation of successfully removing the mT gene. 

This mouse model was originally developed for Cre recombinase, which is an enzyme able to perform site-specific recombination on certain areas of the genome known as “LoxP.” Therefore, if the mT gene is between two LoxP sites, then Cre recombinase is able to cut at both locations and effectively excise the mT gene. 

We take this idea and apply it to CRISPR-Cas9.  By “programming” our CRISPR-Cas9 with a guide RNA (gRNA) able to detect LoxP sites, we hope to obtain mouse organs that has as green as a fluorescence as the organs with Cre recombinase.

This project involves working with mice.  During this past summer, I helped breed mice, wean mice, genotype mice, inject pregnant mice with viral vectors carrying CRISPR-Cas9, and euthanized mice.  We were able to collect the organs of the mice at varying time points and to observe their fluorescence under a stereomicroscope. Finally, I also learned many lab techniques that aided in the data collection of our project, such as removing organs from the fetuses and extracting DNA from these organs.

This project gave me a solid foundation and understanding in gene editing and in the potential of CRISPR-Cas9. This past summer, I was able to apply a lot of the knowledge I’ve gained through my science courses to my work, which gave me a deeper understanding of the course material that I have learned.