Homology-independent targeted integration (HITI) donor cassette DNA (HITI donor DNA)

Homology-independent targeted integration (HITI) donor cassette DNA is a synthetic DNA molecule designed to achieve precise gene insertion into the host genome, particularly in non-dividing cells where traditional homology-directed repair (HDR) is inefficient (Suzuki et al., 2016, Nature). The HITI strategy relies on the Non-Homologous End Joining (NHEJ) pathway, which is active throughout the cell cycle, unlike HDR which is restricted to the S/G2 phases (Yao et al., 2017, Cell Research). The donor cassette is typically engineered with CRISPR/Cas9 target sites flanking the transgene; these sites are identical to the genomic target site but oriented such that correct integration destroys the recognition site, while incorrect (inverted) integration recreates it for re-cleavage (Suzuki et al., 2016). This "self-cleaving" mechanism ensures high-efficiency, directional knock-in of the genetic payload. HITI has shown significant promise in preclinical models for treating monogenic disorders, such as correcting mutations in the MERTK gene for retinitis pigmentosa (Li et al., 2020, Molecular Therapy). This technology represents a major advancement in gene therapy by expanding the range of targetable tissues to include post-mitotic organs like the brain, heart, and eyes.

Other names

HITI donorHomology-independent donor DNANHEJ-mediated donor cassetteHITI template

Facilitates the insertion of a DNA sequence into a specific genomic locus via the Non-Homologous End Joining (NHEJ) repair pathway following double-strand breaks induced by a nuclease.