Expanding the Genome Editing Toolbox for Biomedical Applications

Muhammad Waqas Luqman (Advisor: Fabien Delerue, PhD)

             The development of programmable endonucleases and improved transgene-delivery methods has revolutionized genome editing in mice. One such strategy is CRISPR-READI, which combines adeno-associated virus (AAV)-mediated gene delivery with electroporation of Cas9-RNPs. The AAV delivers transgenes (up to 4.7 kb) as ssDNA to the zygotes, which is followed by electroporation of Cas9-RNPs. This results in improved and precise genome editing. However, a thorough process for the quality control (QC) of the targeted site following this method has yet to be developed, and as a result, unwanted mutations may go undetected.

             To this end, we targeted two mouse genes, Ace2 and Foxg1, via CRISPR-READI, and generated three GM mouse lines. Next, we employed Nanopore-Cas9-targeted sequencing (nCATS) to analyze the targeted sites and observed concatemer formation, along with unintended insertion of AAV’s inverted terminal repeat (ITR) sequences. Both of these bystander mutations were missed by standard genotyping.

              This highlights the need for more thorough validation and development of improved transgene delivery methods. Therefore, we further investigated alternative delivery methods, including DNA origami (DO), elastin-like polypeptides (ELPs), and sperm-mediated delivery. We showed that DO nanostructures were efficiently taken up by zygotes and crossed both the ZP and plasma membrane. In contrast, ELPs and sperm-mediated delivery were ineffective. Overall, these results emphasize the importance of long-read sequencing for QC and support DO as a promising alternative for safer, more reliable gene delivery.

Advisory Committee:

• Fabien Delerue, PhD, Chair
• Richard Behringer, PhD
• George Calin, MD, PhD
• Guillermina Lozano, PhD
• Aimee Stablewski, PhD 

Join via Zoom (Please contat Mr. Luqman for his Zoom meeting info)