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. 2014 Jan 23:4:3831.
doi: 10.1038/srep03831.

BurrH: a new modular DNA binding protein for genome engineering

Alexandre Juillerat  1 , Claudia Bertonati  1 , Gwendoline Dubois  2 , Valérie Guyot  2 , Séverine Thomas  2 , Julien Valton  2 , Marine Beurdeley  2 , George H Silva  2 , Fayza Daboussi  2 , Philippe Duchateau  2
Affiliations

BurrH: a new modular DNA binding protein for genome engineering

Alexandre Juillerat et al. Sci Rep. .

Abstract

The last few years have seen the increasing development of new DNA targeting molecular tools and strategies for precise genome editing. However, opportunities subsist to either improve or expand the current toolbox and further broaden the scope of possible biotechnological applications. Here we report the discovery and the characterization of BurrH, a new modular DNA binding protein from Burkholderia rhizoxinica that is composed of highly polymorphic DNA targeting modules. We also engineered this scaffold to create a new class of designer nucleases that can be used to efficiently induce in vivo targeted mutagenesis and targeted gene insertion at a desired locus.

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Conflict of interest statement

All co-authors are Cellectis employees.

Figures

Figure 1
Figure 1. Strategy for identifying novel modular DNA targeting platforms.
Workflow used to identify new specific and modular DNA binding domains. Three principal features were used as guidelines for a bioinformatic search: modularity, polymorphism and specificity. Four families of databases were queried for different protein features: structure, sequence, localization, function. The outputs of the various databases were cross-referenced and manually inspected. The final filtered results provided the hits to be further characterized experimentally.
Figure 2
Figure 2. Description of a new M3BD (Modular Base-per-Base Binding Domain) scaffolds from Burkholderia rhizoxinica.
(a) Sequence logo of the natural modules of AvrBs3 and BurrH as output by WEBLOGO. (b) Sequence and secondary structure prediction of the first module of BurrH. The sequence is indicated in single letter code. Cylinders represent helices (H). (c) Schematic layout of the organization of AvrBS3 (top) and BurrH (bottom). Colors represent the diversity of the modules.
Figure 3
Figure 3. Engineering of the BurrH scaffold for the generation of target-specific nucleases in S. cerevisiae.
(a) Schematic representation of the BurrH-based nuclease bound to its designed target. All engineered BurrH-based nucleases contained an N-terminal nuclear localization sequence (NLS). (b) Activity of BurrH-based nuclease (bn17421) and AvrBs3-TALE nuclease (TALEN) on their respective homodimeric targets (spacer of 15 bp). Activity was measured using a Single Strand Annealing (SSA) based assay (Supplementary Fig. 2). Error bars denote s.d.; n ≥ 10. (c) Activity of BurrH-based nuclease (bn17421) 2 depending on the nucleotide in position −2, −1 or 0. A set of 20 homodimeric targets, separated by a DNA spacer of 15 bp and containing various combinations of the four bases in positions 0, −1 and −2 was created. Activity of BurrH-based nuclease was monitored as outlined in (b) individually on each target of the set. Each dataset contained at least three individual measurement points. Error bars denote s.d.; n ≥ 4. Control represents the mean value of experiments performed in absence of nuclease expressing vector.
Figure 4
Figure 4. Characterization of BurrH-based nucleases in vivo (293H).
(a) Alignment of the WT genomic sequence and the most predominant events (deletions are indicated by dashes) induced by the BurrH-based nuclease (20 modules, bn21604/21608) at the CAPNS1 locus. Binding sites of the two half BurrH-based nuclease are underlined. (b) Size distribution of the deletion (blue bars) and insertion (red bars) events induced at the CAPNS1 locus by the BurrH-based nuclease composed of 20 modules. (c) Targeted gene insertion frequency determined at the CAPNS1 locus in the presence (+) or absence (-) of the nuclease. Error bars denote s.d.; n = 2.
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References

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