Precise in vivo genome editing via single homology arm donor mediated intron-targeting gene integration for genetic disease correction

Abstract

In vivo genome editing represents a powerful strategy for both understanding basic biology and treating inherited diseases. However, it remains a challenge to develop universal and efficient in vivo genome-editing tools for tissues that comprise diverse cell types in either a dividing or non-dividing state. Here, we describe a versatile in vivo gene knock-in methodology that enables the targeting of a broad range of mutations and cell types through the insertion of a minigene at an intron of the target gene locus using an intracellularly linearized single homology arm donor. As a proof-of-concept, we focused on a mouse model of prematureaging caused by a dominant point mutation, which is difficult to repair using existing in vivo genome-editing tools. Systemic treatment using our new method ameliorated aging-associated phenotypes and extended animal lifespan, thus highlighting the potential of this methodology for a broad range of in vivo genome-editing applications.

Genome Editing for CNS Disorders

“…Several groups have proposed NHEJ-like strategies to overcome this limitation through precise gene editing in non-replicative cells by microhomologymediated end-joining (MMEJ) (Yao et al, 2017b), homologyindependent targeted integration (HITI) (Suzuki et al, 2016), and microhomology-dependent targeted integration (MITI) (Li et al, 2019). Other groups have explored HDR-like mechanisms, such as homology-mediated end joining (HMEJ) (Yao et al, 2017a) and single homology arm donor-mediated intron-targeting integration (SATI) (Suzuki et al, 2019). These techniques have yielded significantly higher rates of gene insertion in postmitotic cells, although the mechanisms involved are not fully understood.…”

CRISPR-Cas systems: Overview, innovations and applications in human disease research and gene therapy

“…In addition, Suzuki et al . repaired G609G mutation in a HGPS mouse model via single homology arm donor mediated intron-targeting gene integration (SATI), which ameliorated aging-associated phenotypes and extended the lifespan of HGPS mice [191] .…”

Methodologies and Challenges for CRISPR/Cas9 Mediated Genome Editing of the Mammalian Brain

“…Recently, an additional knockin approach termed intercellular linearized single homology arm donor mediated intron-targeting integration (SATI) was developed based on the original HITI method (Suzuki et al, 2019). This approach utilizes a donor FIGURE 6 | HDR mediated genome editing using SLENDER to label endogenos proteins in the brain.…”