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

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.

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.

Figure 2

ZFN-Site Results. ZFN-Site output listing the IL2R-γ target sequence, in row 1, and other genomic sequences matching the search criteria in Figure 1. Non-matching bases are shown in red below the correct base. Between each pair of target sequences is a spacer with its genomic sequence shown in blue. The number of nucleotides in the spacer is indicated by the number of green Ns. Each sequence row also lists the number of mismatches, chromosomal location, DNA strand and HTML links to their exact location on ENSEMBL, UCSC, NCBI and NCBI browsers. The link to results in text format provides sequences in the list ordered by increasing number of mismatches.

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.

Figure 4

ZFN-Site returns sites found in previous CCR5 ZFN off-target analysis. The sequences returned by ZFN-Site were matched to the sequences found by Perez et al. For clarity of presentation, the ZFN-Site output was arranged to match the order of Perez et al. ZFN-Site found all the sites found by the unpublished algorithm of Perez et al., thus validating ZFN-Site. We replaced the column containing the genome browser links with a column referencing the order listed in the first column of Perez, et al. Columns detail the human sequences matching the query, the chromosomal coordinates and the strand. The first row consists of the exact match to the CCR5 genomic sequence.

Figure 5

ZFN half-sites and resulting query sequences with 5 or 6 bp spacing. ZFN-Site generates six query sequences based on the two half-sites entered for homo-dimerization, and two different spacings are allowed between the half-sites. The left and right half-sites are listed followed by the resulting query sequences if the half-sites are separated by 5 or 6 bps. Each list includes a query string made of one left and one right half-site (L + R), two left half-sites (L + L) and two right half-sites (R + R). Each of these would need to be searched individually if using BLAST.