Deletion and replacement of long genomic sequences using prime editing

Area of Science:

• Molecular Biology
• Genetics
• Gene Editing Technologies

Background:

• Genomic insertions, duplications, and indels constitute approximately 14% of human pathogenic mutations.
• Current gene-editing methods struggle with accurate and efficient correction of large genomic alterations (>100 bp).

Purpose of the Study:

• To optimize prime editing (PE) tools for precise genomic deletions and replacements.
• To develop a novel method for replacing large genomic fragments without exogenous DNA templates.

Main Methods:

• Conjugation of Cas9 nuclease to reverse transcriptase (PE-Cas9).
• Utilization of two PE guide RNAs (pegRNAs) targeting complementary DNA strands.
• Development of the PE-Cas9-based deletion and repair (PEDAR) method.

Main Results:

• PEDAR enables precise deletion and replacement of genomic fragments from ~1 kb to ~10 kb with desired sequences up to 60 bp.
• PEDAR demonstrated superior performance over other genome-editing methods in reporter systems and at endogenous loci.
• Efficient creation of large and precise genomic alterations was achieved.

Conclusions:

• The PEDAR method precisely removes pathogenic insertions and repairs deletion junctions, restoring gene expression.
• PEDAR successfully corrected a 1.38-kb pathogenic insertion in the Fah gene in a mouse model of tyrosinemia, restoring FAH expression in the liver.