Highly active zinc-finger nucleases by extended modular assembly

Abstract

Zinc-finger nucleases (ZFNs) are important tools for genome engineering. Despite intense interest by many academic groups, the lack of robust noncommercial methods has hindered their widespread use. The modular assembly (MA) of ZFNs from publicly available one-finger archives provides a rapid method to create proteins that can recognize a very broad spectrum of DNA sequences. However, three-and four-finger arrays often fail to produce active nucleases. Efforts to improve the specificity of the one-finger archives have not increased the success rate above 25%, suggesting that the MA method might be inherently inefficient due to its insensitivity to context-dependent effects. Here we present the first systematic study on the effect of array length on ZFN activity. ZFNs composed of six-finger MA arrays produced mutations at 15 of 21 (71%) targeted loci in human and mouse cells. A novel drop-out linker scheme was used to rapidly assess three-to six-finger combinations, demonstrating that shorter arrays could improve activity in some cases. Analysis of 268 array variants revealed that half of MA ZFNs of any array composition that exceed an ab initio B-score cutoff of 15 were active. These results suggest that, when used appropriately, MA ZFNs are able to target more DNA sequences with higher success rates than other current methods.

Global analysis of Drosophila Cys2-His2 zinc finger proteins reveals a multitude of novel recognition motifs and binding determinants

“…Characterized fingers from naturally occurring ZFPs have been successfully utilized as modules to assemble artificial TFs or nucleases for targeted gene disruption (Bae et al 2003;Kim et al 2009Kim et al , 2011. While single fingers-primarily of artificial origin (Segal et al 1999;Dreier et al 2000Dreier et al , 2001Dreier et al , 2005Liu et al 2002;Zhu et al 2011a)--have been the mainstay of archives for the assembly of ZFAs with novel DNA-binding specificity (Liu et al 1997;Carroll et al 2006;Mandell and Barbas 2006;Wright et al 2006;Kim et al 2009;Zhu et al 2011a;Bhakta et al 2013), more recent assembly methods have focused on archives of two-finger modules (Doyon et al 2008;Kim et al 2011;Sander et al 2011;Gupta et al 2012;Zhu et al 2013) to reduce the number of ''novel'' fingerfinger interfaces that are incorporated into the ZFA (Urnov et al 2010). Consequently, we examined the utility of one and two finger Drosophila modules for the creation of ZFAs with novel specificity.…”

A Cut above the Rest: Targeted Genome Editing Technologies in Human Pluripotent Stem Cells

“…This design further enhances the specificity of ZFNs, as dimerization of FokI and thus cleavage are dependent on a longer target. A pair of ZFNs that each contains three zinc finger motifs are sufficient to ensure a unique intended target of an 18-bp sequence by chance in the human genome, although four to six motifs are more commonly used to increase specificity (12). ZFNs have been successfully used to edit the genome of many organisms, including humans (11,13).…”

Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9

“…While this may be a nonissue if an investigator seeks to knock out a gene, since a frameshift introduced anywhere in the early coding sequence of the gene can produce the desired result, it may present challenges if a particular site is required, e.g., to knock in a specific mutation. Since the introduction of the open-source platform, alternative platforms to engineer optimized ZFNs have emerged, each with varying degrees of speed, flexibility in site selection, and success rates (14)(15)(16)(17)(18).…”