Trey Gustafson
Pronouns: He/him/his
Research Mentor(s): Yan Zhang
Research Mentor School/College/Department: Biological Chemistry / Medicine
Program: CG
Session: Session 7 (4:40pm – 5:30pm)
Authors: Trey Gustafson, Renke Tan, Yan Zhang
Abstract
Twin prime editing methods, pioneered by the David R. Liu lab, use two pegRNAs, a reverse transcriptase, and Cas9 nickase to delete and replace segments of DNA. Twin prime editing eliminates the need for double-stranded breaks that incur unintended gene editing outcomes, reducing toxicity to the host cell. While increasing the overall precision of deletions, current twin prime methods only perform deletions up to 800bp. Many human gene editing applications, such as full gene removal, require deletions in the range of 10Kbp. In this project, two pegRNAs were designed to introduce a 10Kbp deletion within the human HPRT1 gene, which causes Lesch-Nyhan syndrome. The two unique pegRNAs, engineered with specific binding sites and sequences, were combined with a prime editor and delivered to HAP1 cells. The HAP1 cells then underwent 6-TG selection for HPRT-1 deficient cells. We confirmed the successful deletion of the intended 10Kbp DNA fragment on the human genome by PCR and sanger sequencing. From these results, we can conclude the potential to use twin prime editing for long-range deletions. Broadly, the ability to successfully perform long-range deletions will greatly expand the gene-editing tool kit and have significant implications on gene therapy.
