Understanding Risks in Gene Editing: Analyzing Prime Editing Outcomes and Large Deletions Compared to CRISPR/Cas9 in iPSCs

• Applicant: Andrea Rossi, Ph.D.
• Subject Area: Human Genetics ; General Genetics and Functional Genome Biology ; Developmental Biology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 559465680

Genome engineering technologies, particularly CRISPR/Cas9, present challenges in achieving precise and safe genetic modifications, which are critical for translational medicine. CRISPR/Cas9 induces double-strand breaks (DSBs) in DNA, which are repaired through non-homologous end joining (NHEJ) or homology-directed repair (HDR). These repair pathways often result in unintended on-target effects, including large deletions, insertions (indels), and structural variants, which can disrupt gene function and lead to genomic instability. While off-target effects have been extensively studied, the occurrence and extent of large deletions at on-target sites remain less understood, posing a risk of misidentifying hemizygous mutations as homozygous when large deletions go undetected. Prime editing (PE) offers a more precise and versatile approach by enabling base substitutions, small insertions, and deletions without generating DSBs, thereby reducing the likelihood of unintended edits. However, despite its precision, there remains a need to systematically assess the potential on-target risks of large deletions and other unwanted outcomes in prime editing. A direct comparison of CRISPR/Cas9 and PE in this context is crucial to better understand the relative risks and advantages of each technology. In this project, we aim to systematically investigate the on-target effects of CRISPR/Cas9 and prime editing in therapeutically relevant induced pluripotent stem cells (iPSCs). By designing and transfecting guide RNAs and prime editing guide RNAs (pegRNAs) into both male and female iPSC lines, we will generate targeted edits. Fluorescence-Activated Cell Sorting (FACS) will be employed to isolate successfully edited cells. Preliminary analysis of editing efficiency and locus integrity will be conducted using digital PCR (dPCR). Comprehensive sequencing using both short-read Illumina (for small indels) and long-read Nanopore platforms (for large deletions up to 10kb) will be performed to thoroughly examine on-target editing outcomes, including large deletions and structural variants. Additionally, we will refine our previously developed software, CRISPRnano, to improve its capability in analyzing prime editing outcomes, particularly focusing on better alignment, visualization, and detection of unintended indels. This research will provide critical insights into the on-target safety profiles of CRISPR/Cas9 and prime editing, informing the development of more accurate and reliable genome editing tools for research and therapeutic applications.

• DFG Programme: Research Grants