Dissertation Defense: Aisha AlJanahi
Candidate Name: Aisha AlJanahi
Major: Biochemistry and Molecular Biology
Advisor: Cynthia Dunbar, Ph.D.
Title: Validation and Long-Term Follow Up of CD33 Off-Targets Predicted in vitro and in silico Using Error Corrected Sequencing in Rhesus Macaques
The programmable nuclease technology CRISPR/Cas9 has revolutionized gene editing in the last decade. Due to the risk of off-target editing, accurate and sensitive methods for off-target characterization are crucial prior to applying CRISPR/Cas9 therapeutically. Here, we utilized a rhesus macaque model to ask whether CIRCLE-Seq (CS), an in vitro off-target prediction method, more accurately identifies OTs compared to in silico prediction (ISP) based solely on genomic sequence comparisons. We use AmpliSeq HD error-corrected sequencing to validate off-target sites predicted by CIRCLE-Seq and in silico prediction for guide RNAs designed against TET2 and CD33 genes. A gRNA targeting TET2 designed using modern algorithms and predicted to have low off-target risk by both in silico prediction and CIRCLE-Seq created no detectable mutations at off-target sites in hematopoietic cells following transplantation, even when applying highly sensitive error-corrected sequencing. In contrast, a CD33 gRNA designed using less robust algorithms with over 10-fold more off-targets sites predicted by both in silico prediction and CIRCLE-Seq resulted in 19 detectable off-target mutations in in blood cells collected from edited animals following transplantation with CRISPR/Cas9-edited HSPCs. A subset of these 19 edited sites was predicted by CIRCLE-Seq only. Cells with off-target editing exhibited no expansion or abnormal behavior in vivo in animals followed for up to 2 years.