TARGET PROJECT 4

Gene Editing of the Beta Globin Gene Using CRISPR/Cas9 and single stranded Oligonucleotides

Investigator:  Natalia Rivera-Torres, Ph.D.

Dr. Natalia Rivera-Torres is a Research Scientist and is credited with elucidating the mechanism of homology directed repair in human cells in a process known as Single-Strand Template Repair (SSTR) or ExACT, EXcision And Corrective Therapy. She received her Ph.D. from the University of Delaware, her dissertation focused on CRISPR Directed Targeted Gene Alteration: Mechanism to Application. Her work has led to a fundamental understanding of how gene repair is carried out in human cells and how this process may vary in a variety of different patient samples. She is now focusing on developing a platform for predicting gene editing outcomes in diverse patient population with Sickle Cell Anemia.

Project Summary:

We have utilized a rapid in vitro gene editing system (figure 1) to determine the capacity of a CD34+ HSPCs cell-free extracts to catalyze pathways of repair. This in vitro system allows for independent assessment of genetic diversity by using different patient samples through the enzymatic activity of ribonucleoprotein particles (CRISPR-Cas9 or CRISPR/Cas12a) to execute site-specific cleavage, with double-stranded plasmid DNA serving as the target, along with a donor template. The CD34+ HSPCs were used to produce a cell lysate (cell free extract) that catalyzed the gene editing reaction. For this system we designed a plasmid that contains 502bp of the HBB gene that has the two regions (G5 and G10) previously studied to induce the sickle cell mutation utilizing CRISPR/Cas9. The oligonucleotide used in these experiments is 72 bases in length that contains the single base pair exchange to create a mismatch to produce the sickle cell sequence. The goal of this study is the use of the cell lysate produced from different donor CD34+ cells will set the base work for determining individual’s capacity to execute gene editing.

Publications:

https://www.ncbi.nlm.nih.gov/myncbi/1HipbIirebzohy/bibliography/public/