# The precision strategy of human genome correction via a set of circular donor DNA and its cleaver

## TL;DR

This paper introduces a new precision genome editing strategy using circular donor DNA and cleavage to replace disease-causing alleles in human cells.

## Contribution

The novel InCDC technique enables high-efficiency, precise crossover-type HR-based gene editing in human cells.

## Key findings

- InCDC generates targeted duplication of circular donor plasmid at the target locus with higher efficiency than linearization techniques.
- The doublet configuration formed by InCDC leads to retention of the designed allele after cleavage.
- Safety distance within the circular donor plasmid is critical to prevent enzymatic exclusion of the designed sequence.

## Abstract

Homologous recombination (HR) corrects a mutational sequence causing a genetic disease by replacing it with the normal sequence to restore a healthy state in humans. A targeted genomic breakage, such as that induced by CRISPR–Cas9, can trigger a copy-paste-type HR event; however, CRISPR–Cas9 more frequently induces imprecise non-homologous end-joining events, leading to one-step multiple knockout products for paralogous genes or homologous alleles, which can be considered a unique advantage. We have established a precision strategy for crossover-type HR-based gene editing, primed by intra-cellular circular donor cleavage (InCDC). The InCDC technique generates targeted duplication of the circular donor plasmid at the target locus in human cells, forming a doublet configuration comprising the donor DNA with the designed sequence and the target DNA with the original sequence, with much higher efficiency than conventional donor linearization techniques. This doublet form leads to the singlet form, resulting in retention of the designed allele. We found that the safety distance within the designed circular donor plasmid and its intra-cellular cleavage was particularly critical to protect a designed sequence from enzymatic exclusion, and we propose that InCDC technology enables precision genome editing, such as the replacement of a genetic disease-causing allele with the correctly designed allele.