Deletion and replacement of long genomic sequences using prime editing

Genomic insertions, duplications and insertion/deletions (indels), which account for similar to 14% of human pathogenic mutations, cannot be accurately or efficiently corrected by current gene-editing methods, especially those that involve larger alterations (>100 base pairs (bp)). Here, we optimize prime editing (PE) tools for creating precise genomic deletions and direct the replacement of a genomic fragment ranging from similar to 1 kilobases (kb) to similar to 10 kb with a desired sequence (up to 60 bp) in the absence of an exogenous DNA template. By conjugating Cas9 nuclease to reverse transcriptase (PE-Cas9) and combining it with two PE guide RNAs (pegRNAs) targeting complementary DNA strands, we achieve precise and specific deletion and repair of target sequences via using this PE-Cas9-based deletion and repair (PEDAR) method. PEDAR outperformed other genome-editing methods in a reporter system and at endogenous loci, efficiently creating large and precise genomic alterations. In a mouse model of tyrosinemia, PEDAR removed a 1.38-kb pathogenic insertion within the Fah gene and precisely repaired the deletion junction to restore FAH expression in liver.

Automated summary

The study optimized prime editing tools for creating precise genomic deletions and direct replacement of a genomic fragment with a desired sequence without the need for an exogenous DNA template. By conjugating Cas9 nuclease to reverse transcriptase and using two PE guide RNAs targeting complementary DNA strands, the PEDAR method achieved precise and specific deletion and repair of target sequences.