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ZFN-site searches genomes for zinc finger nuclease target sites and off-target sites.

Cradick TJ, Ambrosini G, Iseli C, Bucher P, McCaffrey AP - BMC Bioinformatics (2011)

Bottom Line: ZFN-Site facilitates genome searches for possible ZFN cleavage sites based on user-defined stringency limits.ZFN-Site is an improvement over other methods because the FetchGWI search engine uses an indexed search of genome sequences for all ZFN target sites and possible off-target sites matching the half-sites and stringency limits.Therefore, ZFN-Site does not miss potential off-target sites.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Iowa School of Medicine, Department of Internal Medicine, Iowa City, Iowa 52245, USA. tj@alum.mit.edu

ABSTRACT

Background: Zinc Finger Nucleases (ZFNs) are man-made restriction enzymes useful for manipulating genomes by cleaving target DNA sequences. ZFNs allow therapeutic gene correction or creation of genetically modified model organisms. ZFN specificity is not absolute; therefore, it is essential to select ZFN target sites without similar genomic off-target sites. It is important to assay for off-target cleavage events at sites similar to the target sequence.

Results: ZFN-Site is a web interface that searches multiple genomes for ZFN off-target sites. Queries can be based on the target sequence or can be expanded using degenerate specificity to account for known ZFN binding preferences. ZFN off-target sites are outputted with links to genome browsers, facilitating off-target cleavage site screening. We verified ZFN-Site using previously published ZFN half-sites and located their target sites and their previously described off-target sites. While we have tailored this tool to ZFNs, ZFN-Site can also be used to find potential off-target sites for other nucleases, such as TALE nucleases.

Conclusions: ZFN-Site facilitates genome searches for possible ZFN cleavage sites based on user-defined stringency limits. ZFN-Site is an improvement over other methods because the FetchGWI search engine uses an indexed search of genome sequences for all ZFN target sites and possible off-target sites matching the half-sites and stringency limits. Therefore, ZFN-Site does not miss potential off-target sites.

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Benchmarking ZFN-Site against a published CCR5 ZFN off-target analysis. Previously, Perez et al. used SELEX to determine the relaxed specificity of a ZFN pair targeting the CCR5 gene and used this data to scan the genome. We scanned the human genome with ZFN-Site, configured as shown, using the CCR5 ZFN half-sites from Perez et al. with ambiguities matching their SELEX data. The bases allowing substitutions are shown in lower case letters. ZFN-Site found each site they listed, paired with their results in Figure 4.
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Figure 3: Benchmarking ZFN-Site against a published CCR5 ZFN off-target analysis. Previously, Perez et al. used SELEX to determine the relaxed specificity of a ZFN pair targeting the CCR5 gene and used this data to scan the genome. We scanned the human genome with ZFN-Site, configured as shown, using the CCR5 ZFN half-sites from Perez et al. with ambiguities matching their SELEX data. The bases allowing substitutions are shown in lower case letters. ZFN-Site found each site they listed, paired with their results in Figure 4.

Mentions: More specific information comes from binding studies of full ZFPs or ZFNs using SELEX. Searches based on experimentally determined specificity are more informative than searches with increased mismatches. If there is SELEX or similar data describing each ZFN's binding specificity, it is also entered in 5' to 3' orientation using standard IUPAC ambiguity codes (as in Figure 3). This allows relaxed specificity searches. For example, a nucleotide in a half-site that can be bound if it is either G or T can be entered as a K. Any non-specified position can be represented by an N (N=A, C, G or T). If scanning with two mismatches, the pair of half-sites should contain less than three ambiguities to prevent computational stalling (see above).


ZFN-site searches genomes for zinc finger nuclease target sites and off-target sites.

Cradick TJ, Ambrosini G, Iseli C, Bucher P, McCaffrey AP - BMC Bioinformatics (2011)

Benchmarking ZFN-Site against a published CCR5 ZFN off-target analysis. Previously, Perez et al. used SELEX to determine the relaxed specificity of a ZFN pair targeting the CCR5 gene and used this data to scan the genome. We scanned the human genome with ZFN-Site, configured as shown, using the CCR5 ZFN half-sites from Perez et al. with ambiguities matching their SELEX data. The bases allowing substitutions are shown in lower case letters. ZFN-Site found each site they listed, paired with their results in Figure 4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113941&req=5

Figure 3: Benchmarking ZFN-Site against a published CCR5 ZFN off-target analysis. Previously, Perez et al. used SELEX to determine the relaxed specificity of a ZFN pair targeting the CCR5 gene and used this data to scan the genome. We scanned the human genome with ZFN-Site, configured as shown, using the CCR5 ZFN half-sites from Perez et al. with ambiguities matching their SELEX data. The bases allowing substitutions are shown in lower case letters. ZFN-Site found each site they listed, paired with their results in Figure 4.
Mentions: More specific information comes from binding studies of full ZFPs or ZFNs using SELEX. Searches based on experimentally determined specificity are more informative than searches with increased mismatches. If there is SELEX or similar data describing each ZFN's binding specificity, it is also entered in 5' to 3' orientation using standard IUPAC ambiguity codes (as in Figure 3). This allows relaxed specificity searches. For example, a nucleotide in a half-site that can be bound if it is either G or T can be entered as a K. Any non-specified position can be represented by an N (N=A, C, G or T). If scanning with two mismatches, the pair of half-sites should contain less than three ambiguities to prevent computational stalling (see above).

Bottom Line: ZFN-Site facilitates genome searches for possible ZFN cleavage sites based on user-defined stringency limits.ZFN-Site is an improvement over other methods because the FetchGWI search engine uses an indexed search of genome sequences for all ZFN target sites and possible off-target sites matching the half-sites and stringency limits.Therefore, ZFN-Site does not miss potential off-target sites.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Iowa School of Medicine, Department of Internal Medicine, Iowa City, Iowa 52245, USA. tj@alum.mit.edu

ABSTRACT

Background: Zinc Finger Nucleases (ZFNs) are man-made restriction enzymes useful for manipulating genomes by cleaving target DNA sequences. ZFNs allow therapeutic gene correction or creation of genetically modified model organisms. ZFN specificity is not absolute; therefore, it is essential to select ZFN target sites without similar genomic off-target sites. It is important to assay for off-target cleavage events at sites similar to the target sequence.

Results: ZFN-Site is a web interface that searches multiple genomes for ZFN off-target sites. Queries can be based on the target sequence or can be expanded using degenerate specificity to account for known ZFN binding preferences. ZFN off-target sites are outputted with links to genome browsers, facilitating off-target cleavage site screening. We verified ZFN-Site using previously published ZFN half-sites and located their target sites and their previously described off-target sites. While we have tailored this tool to ZFNs, ZFN-Site can also be used to find potential off-target sites for other nucleases, such as TALE nucleases.

Conclusions: ZFN-Site facilitates genome searches for possible ZFN cleavage sites based on user-defined stringency limits. ZFN-Site is an improvement over other methods because the FetchGWI search engine uses an indexed search of genome sequences for all ZFN target sites and possible off-target sites matching the half-sites and stringency limits. Therefore, ZFN-Site does not miss potential off-target sites.

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