Multiple copies of a linear donor fragment released in situ from a vector improve the efficiency of zinc-finger nuclease-mediated genome editing

Abstract

Homologous recombination (HR) is a strategy for genetic correction. The efficiency of HR can be increased by creating a targeted double-strand break (DSB) via zinc-finger nucleases (ZFNs) and/or by introducing linear donor DNA intracellularly. Some studies have suggested that increased copy numbers of linear donor DNA may further improve HR efficiency. However, the introduction of multiple copies of a linear donor fragment remains a challenge, particularly in cell types with low transfection efficiency. In this study, we developed a vector that carries tandem repeats of a donor fragment, with each repeat flanked by ZFN target sequence fragments (TSFs). The cleavage of the flanking TSF sequence by ZFN would lead to the release of multiple linear fragment. We demonstrated that this novel vector carrying five copies of a linearizable donor fragment, when co-transfected with a ZFN-expressing vector in 293 cells, showed improved HR efficiency about 30 times, as compared with vector carry nonlinearizable donor. For the application of gene therapy, we then introduced this system into an adenoviral vector, which also revealed markedly improved ZFN-mediated HR efficiency in cells. The novel strategies presented here have the potential to promote the application of ZFNs in both basic research and disease therapy.

CRISPR-Mediated Integration of Large Gene Cassettes Using AAV Donor Vectors

“…While other viral vectors with larger carrying capacity have been used to deliver HR templates, e.g. gutless adenoviral vectors and integration defective lentiviral vectors (IDLV) (Knipping et al, 2017, Hoban et al, 2016, Holkers et al, 2014, Zhang et al, 2014a, Genovese et al, 2014, Zhang et al, 2014b), AAV is currently the vector platform of choice for gene editing in primary T cells and HSPCs since it supports high rates of homologous recombination (Sather et al, 2015). However, in other cell types, different viral vectors may be superior in donor template delivery.…”

CRISPR/Cas9 Mediated Therapeutic Approach in Huntington’s Disease

“…Nuclease domain of endonuclease Fok-1 is known as the second part. Nuclease domains may form double chain breakage after dimerization of two ZFN subunits close together, and these nuclease domains are the same [ 37 , 38 ]. The transcriptional repressor protein or specific nuclease is connected to the DNA-binding element consisting of a series of multiple zinc-finger peptides.…”

One-Vector System for Multiplexed CRISPR/Cas9 against Hepatitis B Virus cccDNA Utilizing High-Capacity Adenoviral Vectors

“…For instance, ZFNs were delivered by adenoviral vectors. 7 , 11 , 12 , 13 Similarly, intact TALEN sequences could be maintained as individual expression cassettes in first- and second-generation adenoviral vectors, 14 , 15 , 16 as well as a complete TALEN pair in HCAdV. 7 , 17 The CRISPR/Cas9 system was packed in early generation adenoviral vectors, too, including the expression cassettes of Cas9 and guide RNA (gRNA) apart 18 or in combination.…”