Rapid “Open-Source” Engineering of Customized Zinc-Finger Nucleases for Highly Efficient Gene Modification

Abstract

Summary Custom-made zinc-finger nucleases (ZFNs) can induce targeted genome modifications with high efficiency in cell types including Drosophila, C. elegans, plants, and humans. A bottleneck in the application of ZFN technology has been the generation of highly specific engineered zinc-finger arrays. Here we describe OPEN (Oligomerized Pool ENgineering), a rapid, publicly available strategy for constructing multi-finger arrays, which we show is more effective than the previously published modular assembly method. We used OPEN to construct 37 highly active ZFN pairs which induced targeted alterations with high efficiencies (1 to 50%) at 11 different target sites located within three endogenous human genes (VEGF-A, HoxB13, CFTR), an endogenous plant gene (tobacco SuRA), and a chromosomally-integrated EGFP reporter gene. In summary, OPEN provides an “open-source” method for rapidly engineering highly active zinc-finger arrays, thereby enabling broader practice, development, and application of ZFN technology for biological research and gene therapy.

Reply to “Genome editing with modularly assembled zinc-finger nucleases”

“…In addition, we found in a different direct comparison of OPEN and modular assembly at five different target sites in the EGFP gene that OPEN ZFNs were active at four sites whereas modularly assembled ZFNs showed activity at only one site. 4 Furthermore, the OPEN ZFNs outperformed the modularly assembled ZFNs at this one site. We believe that the higher activity and success rate of OPEN ZFNs is most likely due to the method's explicit consideration of the well-established context-dependent behavior of zinc fingers, 1, 4 a parameter that is largely ignored by modular assembly.…”

“…4 Kim et al contend that our recent report2 shows that "modularly assembled ZFNs…outperformed OPEN ZFNs in terms of mutation frequencies" and that "each method [modular assembly and OPEN] gave rise to successful genome modification at one out of four target sites." However, the modularly assembled and OPEN ZFNs in our study were designed to recognize different DNA target sites and therefore Kim et al's conclusion is based on an indirect comparison.…”

“…In addition, we found in a different direct comparison of OPEN and modular assembly at five different target sites in the EGFP gene that OPEN ZFNs were active at four sites whereas modularly assembled ZFNs showed activity at only one site. 4 Furthermore, the OPEN ZFNs outperformed the modularly assembled ZFNs at this one site. We believe that the Correspondence should be addressed to: J. Keith Joung (jjoung@partners.org), Daniel F. Voytas (voytas@umn.edu), and Toni Cathomen (cathomen.toni@mh-hannover.de).…”

Crossing the germline: CRISPR-Cas9 and our responsibility as reproductive endocrinology and infertility physicians

“…Importantly, how often does the desired mutation find its way into the target genome? The targeting efficiency of the CRISPR-Cas9 system appears superior to other gene-editing techniques such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), although these techniques set a low bar to reach with efficiencies in human cells ranging from 1 to 50% [7][8][9]. Assuredly, this would be expected to improve with further research, but such inefficiency can produce mosaic cell lines within the embryo, in which some of the cells have been restored to wild type and others remain unrepaired.…”

DNA sense-and-respond protein modules for mammalian cells

“…1 and Supplementary Note 1) 1,5 . To generate two hexadactyl ZF sensors of high affinity and specificity, we first applied an extended linker that permits finger multimerization without excessive DNA strain, and merged two pairs of the tridactyl ZFs 17, 18 .…”