Programming large target genomic deletion and concurrent insertion via a prime editing-based method: PEDAR

Abstract

Genomic insertions, duplications, and insertion/deletions (indels) account for ~14% of human pathogenic mutations. Current gene editing methods cannot accurately or efficiently correct these abnormal genomic rearrangements, especially larger alterations (>100 bp). Thus, developing a method to accurately delete insertions/duplications and repair the deletion junction could improve the scope of gene therapies. Here, we engineer a novel gene editor, PE-Cas9, by conjugating Cas9 nuclease to reverse transcriptase. Combined with two prime editing guide RNAs (pegRNAs) targeting complementary DNA strands, PE-Cas9 can direct the replacement of a genomic fragment, ranging from to ~1-kb to >10-kb, with a desired sequence at the target site without requiring an exogenous DNA template. In a reporter cell line, this PE-Cas9-based deletion and repair (PEDAR) method restored mCherry expression through in-frame deletion of a disrupted GFP sequence. We further show that PEDAR efficiency could be enhanced by using pegRNAs with high cleavage activity or increasing transfection efficiency. In tyrosinemia mice, PEDAR removed a 1.38-kb pathogenic insertion within the Fah gene and precisely repaired the deletion junction to restore FAH expression in liver. This study highlights PEDAR as a tool for correcting pathogenic mutations.

Comprehending the evolution of gene editing platforms for crop trait improvement

“…This strategy enhanced the editing efficiency to introduce point mutations three-fold (Anzalone et al, 2019). With the use of the same protospacer, off-target instances were found much lower for PEs in comparison to the use of Cas9 (Jiang et al, 2021;Jiang et al, 2022). The increased efficiency of the prime editor is attributed to multiple DNA hybridization events that occur with the use of PEs.…”

“…Next, hybridization occurs between the target sequence in the genomic DNA and the PBS of the pegRNA, adding to the sequence specificity of the system. Finally, the target DNA also hybridizes with the edited DNA, which further adds another layer of sequence specificity to the system ( Jiang et al, 2021 ; Jiang et al, 2022 ). On the contrary, in a regular CRISPR/Cas9 system, only one step of hybridization occurs between the sgRNA and the target genomic DNA occurs ( Jiang et al, 2022 ; Zhuang et al, 2022 ).…”

Gene editing and its applications in biomedicine

“…Although multiple guides can be employed to direct Cas nucleases to induce deletions or inversions of large DNA fragments (Maeder et al, 2019), undesired byproducts occur, increasing safety risks. It is worth noting that several works reported on bioRixv repurposed the prime editor to delete large DNA fragments using dual peg-sgRNAs (Choi et al, 2021;Jiang et al, 2021). However, the efficacy of this approach needs to be improved.…”