Unexpected failure rates for modular assembly of engineered zinc fingers

Ramirez, Cherie L.; Foley, Jason; Wright, David; Müller-Lerch, Felix; Rahman, Shamim H.; Cornu, Tatjana I.; Winfrey, Ronnie J.; Sander, Jeffry D.; Fu, Fengli; Townsend, Jeffrey A.; Cathomen, Toni; Voytas, Daniel F.; Joung, Julia

doi:10.1038/nmeth0508-374

Cited by 374 publications

(299 citation statements)

References 6 publications

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“…However placing this finger at the N terminus leads to a significant diminution of its sequence specificity. This observation is consistent with previous observations of nonadditive behavior of these modules (34) and recent reports that the predictability of sequence recognition by engineered ZFPs is less than desired (41). Taken together, SSLs show that hairpin polyamides are among the most specific DNA binders.…”

Section: Resultssupporting

confidence: 92%

Specificity landscapes of DNA binding molecules elucidate biological function

Carlson

Warren

Hauschild

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

151

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Evaluating the specificity spectra of DNA binding molecules is a nontrivial challenge that hinders the ability to decipher gene regulatory networks or engineer molecules that act on genomes.Here we compare the DNA sequence specificities for different classes of proteins and engineered DNA binding molecules across the entire sequence space. These high-content data are visualized and interpreted using an interactive "specificity landscape" which simultaneously displays the affinity and specificity of a million-plus DNA sequences. Contrary to expectation, specificity landscapes reveal that synthetic DNA ligands match, and often surpass, the specificities of eukaryotic DNA binding proteins. The landscapes also identify differential specificity constraints imposed by diverse structural folds of natural and synthetic DNA binders. Importantly, the sequence context of a binding site significantly influences binding energetics, and utilizing the full contextual information permits greater accuracy in annotating regulatory elements within a given genome. Assigning such context-dependent binding values to every DNA sequence across the genome yields predictive genome-wide binding landscapes (genomescapes). A genomescape of a synthetic DNA binding molecule provided insight into its differential regulatory activity in cultured cells. The approach we describe will accelerate the creation of precision-tailored DNA therapeutics and uncover principles that govern sequence-specificity of DNA binding molecules.

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Section: Resultssupporting

confidence: 92%

Specificity landscapes of DNA binding molecules elucidate biological function

Carlson

Warren

Hauschild

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

151

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“…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.…”

Section: To the Editormentioning

confidence: 99%

“…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).…”

Section: To the Editormentioning

confidence: 99%

“…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."…”

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confidence: 99%

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confidence: 99%

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

2010

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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

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Engineering Designer Nucleases with Customized Cleavage Specificities

Sander

Maeder

Joung

2011

CP Molecular Biology

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Engineered designer nucleases can be used to efficiently modify genomic sequence in a wide variety of model organisms and cell types. Zinc finger nucleases (ZFNs), consisting of an engineered zinc finger array fused to a non-specific cleavage domain, have been extensively used to modify a broad range of endogenous genes. Here we describe protocols for engineering ZFNs targeted to specific gene sequences of interest using the Context-Dependent Assembly (CoDA) method.

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Unexpected failure rates for modular assembly of engineered zinc fingers

Cited by 374 publications

References 6 publications

Specificity landscapes of DNA binding molecules elucidate biological function

Specificity landscapes of DNA binding molecules elucidate biological function

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

Engineering Designer Nucleases with Customized Cleavage Specificities

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