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

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.

Origination of Ds Elements From Ac Elements in Maize: Evidence for Rare Repair Synthesis at the Site of Ac Excision

Although it has been known for some time that the maize transposon Ac can mutate to Ds by undergoing internal deletions, the mechanism by which these mutations arise has remained conjectural. To gain further insight into this mechanism in maize we have studied a series of Ds elements that originated de novo from Ac elements at known locations in the genome. We present evidence that new, internally deleted Ds elements can arise at the Ac donor site when Ac transposes to another site in the genome. However, internal deletions are rare relative to Ac excision footprints, the predominant products of Ac transposition. We have characterized the deletion junctions in five new Ds elements. Short direct repeats of variable length occur adjacent to the deletion junction in three of the five Ds derivatives. In the remaining two, extra sequences or filler DNA is inserted at the junction. The filler DNAs are identical to sequences found close to the junction in the Ac DNA, where they are flanked by the same sequences that flank the filler DNA in the deletion. These findings are explained most simply by a mechanism involving error-prone DNA replication as an occasional alternative to end-joining in the repair of Ac-generated double-strand breaks.

Recent appearance and molecular characterization of mitochondrial DNA deletions within a defined nematode pedigree.

The mitochondrial genome of Romanomermis culicivorax, a parasitic nematode of mosquitoes, contains an amplified 3.0-kilobase (kb) locus organized as direct repeats and as noncontiguous, inverted copies. These amplified sequences are actively undergoing rearrangement. One recent event has resulted in a 1133-base pair (bp) deletion located entirely within a single amplified segment. The deletion junction occurs between two imperfect 58-bp repeats, implicating strand pairing in this alteration. A second event has generated mitochondrial DNA (mtDNA) forms differing by a single, intact 3.0-kb repeating unit. By analyzing molecules derived from independently reared subcultures, it appears these new mtDNA forms arose within the last 170 nematode generations. Our results indicate that the occurrence and selection of novel animal mitochondrial genomes can now be studied in this experimentally manipulable nematode system.