LAHEDES: the LAGLIDADG homing endonuclease database and engineering server

Abstract

LAGLIDADG homing endonucleases (LHEs) are DNA cleaving enzymes, also termed 'meganucleases' that are employed as gene-targeting reagents. This use of LHEs requires that their DNA specificity be altered to match sequences in genomic targets. The choice of the most appropriate LHE to target a particular gene is facilitated by the growing number of such enzymes with well-characterized activities and structures. 'LAHEDES' (The LAGLIDADG Homing Endonuclease Database and Engineering Server) provides both an online archive of LHEs with validated DNA cleavage specificities and DNA-binding interactions, as well as a tool for the identification of DNA sequences that might be targeted by various LHEs. Searches can be performed using four separate scoring algorithms and user-defined choices of LHE scaffolds. The webserver subsequently provides information regarding clusters of amino acids that should be interrogated during engineering and selection experiments. The webserver is fully open access and can be found at http://homingendonuclease.net.

Figure 1.

The specificity profile (PWM) for the I-AniI LHE. Specificity profiles, reported as PWMs that have been experimentally determined for a number of LHEs can be entered individually and are available on the site as part of the browser functionality. These matrices are displayed in graphical format as a sequence logo plot (left) and in tabular format. The ability of the enzyme to cleave DNA targets that contain a given nucleotide at a specific position within the target site is indicated by letter height, and positions with greater overall information content are more specifically recognized. The profile displayed in this figure was determined in a series of biochemical experiments where the cleavability of target site variants was individually measured (34).

Figure 2.

Genomic search results. The human monoamine oxidase B (MAOB) gene was searched for potential target sites matches using the I-OnuI LHE and either the ‘Identity’ algorithm (a) or the ‘Modular Engineerability’ algorithm (b). For both searches, a list of best matches is shown, together with their position in the query sequence, orientation (forward or reverse) and scores. The top scoring hit in the ‘Identity’ Search (Panel A, top MAOB sequence) was targeted for cleavage at the endogenous chromosomal MAOB locus by an engineered version of I-OnuI (20) (listed as ‘I-OnuI(E2)’ in the endonuclease browser, see Figure 1). Throughout the target site hits, mismatches are shown as lower base bases. In the output from a Module Search, each score is linked to a more detailed view (c) illustrating how well each DNA module scores using an ‘engineerability’ matrix for I-OnuI. Modules are represented by individual bars; those that score favorably in a given target are colored blue, while those that do not score as favorably are colored red. Nucleotides that directly match the wild type target are indicated by vertical bars. The engineerability scoring scale (0 to 10; 10 is best) is shown to the right. Clicking on individual bases or modular bars in the search output provides links to lists of amino acids (d) and (if they are available) mutations in the protein that are associated with recognition at those positions in the target.