Here we demonstrated the feasibility of a single homology arm design for CRISPR/Cas9-targeted insertion of a long DNA fragment into a mammalian genome. This strategy simplified the design and cloning procedure of the donor construct by using only a single, short homology arm.

In this case, CRISPR/Cas9 is designed to cleave both the targeted genomic locus and transgene donor vector that contains long homology arms (600–900 bp each homology arm) . This HMEJ-based strategy provides a higher editing efficiency and better fidelity than MMEJ, particularly in non-dividing cells and adult animals 37 .

May 19, 2017 · Here we devise a homology-mediated end joining (HMEJ)-based strategy, using CRISPR/Cas9-mediated cleavage of both transgene donor vector that contains guide RNA target sites and ∼ 800 bp of...

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) technology has brought rapid progress in mammalian genome editing (adding, disrupting or changing the sequence of specific sites) by increasing the frequency of targeted events.

Dec 10, 2024 · CRISPR-Cas9-mediated homology-directed repair (HDR) is a versatile platform for creating precise site-specific DNA insertions, deletions, and substitutions. These precise edits are made possible through the use of exogenous donor templates that carry the desired sequence.

Cas9 will only cleave a given locus if the gRNA spacer sequence shares sufficient homology with the target DNA. Once the Cas9-gRNA complex binds a putative DNA target, the seed sequence (8–10 bases at the 3′ end of the gRNA targeting sequence) will begin to anneal to the target DNA.

We recommend the use of long homology arm targeting constructs for accurate and efficient complex genome engineering, particularly when combined with the simplifying advantages of using just one Cas9 cleavage at the genome target site.

Dec 13, 2023 · Most DNA donor molecules require homology arms (1 kb), a promoter (0.5–1 kb) and a polyadenylation sequence (100–250 bp), thereby restraining transgene size to 1.75–3.4 kb and impeding their...

Objective: CRISPR/Cas9 technology provides a powerful tool for targeted modification of genomes. In this system, a donor DNA harboring two flanking homology arms is mostly used for targeted insertion of long exogenous DNA.

Homology directed repair and genome engineering. CRISPR-Cas9 has revolutionized the genome engineering world and made targeted modifications feasible and even easy. This targeted-break technology coupled with HDR is a powerful site-specific knock-in tool that can be harnessed by researchers for many applications.