Deletion and replacement of long genomic sequences using prime editing

• Published in: Nature biotechnology Vol. 40; no. 2; pp. 227 - 234
• Main Authors: Jiang, Tingting, Zhang, Xiao-Ou, Weng, Zhiping, Xue, Wen
• Format: Journal Article
• Language: English
• Published: United States Nature Publishing Group 01.02.2022
• Subjects: Animals; Complementary DNA; CRISPR; CRISPR-Cas Systems - genetics; Deoxyribonucleic acid; DNA; DNA sequencing; Efficiency; Endonucleases - genetics; FAH gene; Gene deletion; Gene Editing - methods; Genetic modification; Genome; Genome editing; Genomes; Genomics; Insertion; Medical schools; Methods; Mice; Mutation; Nuclease; Nucleotide sequence; Nucleotide sequencing; Repair; RNA, Guide, CRISPR-Cas Systems; RNA-directed DNA polymerase; United States
• ISSN: 1087-0156; 1546-1696
• DOI: 10.1038/s41587-021-01026-y

Genomic insertions, duplications and insertion/deletions (indels), which account for ~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 ~1 kilobases (kb) 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.