Engineered Zinc Finger Nucleases for Targeted Genome Editing

Abstract

Zinc fi nger nucleases (ZFNs) are arti fi cial proteins consisting of engineered zinc fi nger proteins fused to the FokI endonuclease domain. These nucleases bind to speci fi c DNA recognition sites and introduce double-strand breaks (DSBs). Repair of these DSBs by normal cellular processes can be exploited to either disrupt genes or signi fi cantly increase the frequency of homologous recombination with a user-de fi ned repair template. Several platforms have been developed that enable engineering of zinc fi nger proteins that bind with high af fi nity and speci fi city to 9-18 bp target DNA sequences. ZFNs have already been used in basic research, most prominently with the development of methods to ef fi ciently modify various model organisms and cell lines.

Creating cell and animal models of human disease by genome editing using CRISPR/Cas9

“…These studies showed that, in this method, every zinc‐finger domain is affected by the neighboring domain, which also influences the recognition of target sequences by that domain. By changing the number of subsites from two or three to a more variant number, a significant reduction in the DNA‐binding efficiency of ZFN arrays was observed, which confirms the low efficiency of modular assembly . Further studies were conducted aiming to overcome this issue, although the majority required a lot of time, and it was an extremely difficult task to begin with.…”

“…These nucleases were made artificially via genetic engineering by connecting the DNA‐binding domain of ZFPs to the catalytic domain of Fok I endonucleases with the aim of reducing unwanted side‐effects. ZFNs were designed to target genome‐specific sequences with high efficiency . After a ZFN identifies the suitable genomic locus and cleaves it, the HDR system applies the relative changes in that location.…”