Abstract
BackgroundZinc 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.ResultsZFN-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.ConclusionsZFN-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.
Highlights
Zinc Finger Nucleases (ZFNs) are man-made restriction enzymes useful for manipulating genomes by cleaving target DNA sequences
ZFNs consist of the sequence-independent FokI nuclease domain fused to zinc finger proteins (ZFPs)
Perez et al looked for off-target cleavage by a pair of ZFNs specific for the gene coding for human C-C chemokine receptor type 5 (CCR5)
Summary
ZFN-Site was validated by comparing our results to a previously published study by Perez et al [7]. Multiple BLAST searches sometimes accomplish the same function as ZFN-Site if one inputs all possible permutations of homo/heterodimers, spacings and relaxed specificities. The BLAST search for the right homo-dimer pair with six base spacing failed to return two sequences found by Perez et al and ZFN-Site (numbers 10 and 11). This search returned 474 genomic sequences, many of which were too dissimilar to be likely off-target sites.
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