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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.

Show MeSH
ZFN-Site genome scan using Basic Target Search. ZFN-Site search for Sequence 1 using the half-sites described in the text, which are the ZFN target sites found in IL2R-γ [1]. The inputs are set to search the human genome allowing five and six base pair spacing, two mismatches and homo and hetero-dimerization of the half-sites.
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Figure 1: ZFN-Site genome scan using Basic Target Search. ZFN-Site search for Sequence 1 using the half-sites described in the text, which are the ZFN target sites found in IL2R-γ [1]. The inputs are set to search the human genome allowing five and six base pair spacing, two mismatches and homo and hetero-dimerization of the half-sites.

Mentions: Half-sites are entered without spaces, 5' to 3', as they occur on the opposite strand of a ZFN target. The following sequence is an example of the top DNA strand of a three finger ZFN pair target site: 5'-CGGAGCCGCTTTaacccACTCTGTGGAAG-3'[3]. The right ZFN half-site is underlined and should be entered into the program 5'-3' as ACTCTGTGGAAG. The left ZFN half-site is the reverse complement of the bold sequence and should be entered 5'-3' as AAAGCGGCTCCG (Figure 1).


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)

ZFN-Site genome scan using Basic Target Search. ZFN-Site search for Sequence 1 using the half-sites described in the text, which are the ZFN target sites found in IL2R-γ [1]. The inputs are set to search the human genome allowing five and six base pair spacing, two mismatches and homo and hetero-dimerization of the half-sites.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: ZFN-Site genome scan using Basic Target Search. ZFN-Site search for Sequence 1 using the half-sites described in the text, which are the ZFN target sites found in IL2R-γ [1]. The inputs are set to search the human genome allowing five and six base pair spacing, two mismatches and homo and hetero-dimerization of the half-sites.
Mentions: Half-sites are entered without spaces, 5' to 3', as they occur on the opposite strand of a ZFN target. The following sequence is an example of the top DNA strand of a three finger ZFN pair target site: 5'-CGGAGCCGCTTTaacccACTCTGTGGAAG-3'[3]. The right ZFN half-site is underlined and should be entered into the program 5'-3' as ACTCTGTGGAAG. The left ZFN half-site is the reverse complement of the bold sequence and should be entered 5'-3' as AAAGCGGCTCCG (Figure 1).

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.

Show MeSH