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

Abstract

The publications cited by Kim et al. describing successful construction of ZFNs by modular assembly only further support our original conclusion that this method has a high failure rate for engineering functional zinc finger arrays. 1 Two of the three reports cited provide data that enable calculation of failure rates for modular assembly. 2,3 Although it is true that modular assembly yielded ZFNs for ~25% of the DNA sites targeted, failure rates measured instead by the number of zinc finger proteins tested are remarkably consistent with those reported in our original Correspondence. 1 For example, at the human CCR5 gene, Kim et al. screened 315 pairs of ZFNs for activity; 3 this large-scale effort yielded only a small number of functional ZFN pairs (93.3% failure rate for ZFN pairs tested). Similarly, for the tobacco SuRB gene, 2 we tested 32 zinc finger arrays in vitro but identified only three with functional activity (91.6% failure rate for zinc finger arrays tested). These data are consistent with our original predicted failure rates of ~94% and ~76% for modularly assembled ZFN pairs and zinc finger arrays, respectively. 1 We believe that failure rates measured by numbers of zinc finger arrays or ZFN pairs tested rather than by numbers of DNA sites targeted are more relevant statistics for potential ZFN users because these influence how many proteins must be modularly assembled and tested for each potential site.We also respectfully disagree with various statements Kim et al. make regarding Oligomerized Pool ENgineering (OPEN), a selection-based method for engineering zinc finger arrays. 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. For the single site where direct comparison was possible, only the OPEN approach yielded functional ZFNs. 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). NIH Public Access

Targeted Mutagenesis of Duplicated Genes in Soybean with Zinc-Finger Nucleases

“…Several strategies have been employed for the design and construction of ZFNs including modular assembly, oligomerized pool engineering (OPEN), and context-dependent assembly (CoDA; Wright et al, 2006;Maeder et al, 2009;Joung et al, 2010;Kim et al, 2010;Sander et al, 2011). CoDA is the most recent platform developed by the Zinc Finger Consortium and has the advantage in that it is rapid and easy to perform because it does not require labor-intensive selection methods.…”

Genome Engineering With Zinc-Finger Nucleases

“…In fact, a substantial proportion of ZFN pairs fail, whether they are produced by design or selection (Ramirez et al 2008;Joung et al 2010;Kim et al 2010). Even scientists at Sangamo Biosciences and Sigma-Aldrich, who have access to the largest and best-characterized archive of ZFs, make multiple pairs for sequences within a single target gene and test them extensively.…”

Zinc-Finger Nucleases for Somatic Gene Therapy: The Next Frontier

“…1D) (Cornu et al, 2008). Different platforms are available to engineer target-specific zinc-finger arrays Carroll et al, 2006;Mandell and Barbas, 2006;Maeder et al, 2008;Meng et al, 2008;Sander et al, 2011), and the strengths and weaknesses of some of these platforms have been discussed elsewhere (Cathomen and Joung, 2008;Ramirez et al, 2008;Joung et al, 2010;Kim et al, 2010). Second, FokI cleavage domains with altered dimerization specificities have been created.…”