BuD: Another Tunable DNA-Binding Domain for Genome Modification

Tunable DNA-binding domains with high specificity remain important tools for constructing genome modifications even with the current popularity of the CRISPR/Cas9 system.

Rhizopus nigricans – black bread mold

Stella at al (2014) describe their analysis of a DNA-binding protein, BurrH, from the symbiotic bacterium Burkholderia rhizoxinica that is found in the cytosol of the microspores of the fungus Rhizopus.

BurrH contains a DNA-binding domain (BuD) with a central region composed of 19 repeats of a helix-loop-helix modular domain. The BuD identifies the DNA target by a single residue-to-nucleotide code. This allows for redesigning or tuning the protein in a way that is reminiscent of TALEs .

The BuD repeats present some new amino acid-nucleotide interactions as well as those reported for TALEs.

Stella et al (2014) perform some basic biophysical analysis of the BuD as well as reporting its crystal structure both with and without a DNA oligo with its target sequence.  They also demonstrate BuD’s tunability and utility as a genome editing tool.

BuD Crystal Structure with and without DNA

BuD Crystal Structure with and without DNA, (from Stella et al. 2014)

BuD nucleases (BuDN) were tuned to a target to which a TALEN was also tuned and their performance compared. The nuclease activity was similar but BuDN was much less tolerant of target site variants than the TALEN, suggesting more specificity.

The BuD arrays are different from TALENs in that they are the first modular helix-loop-helix domains containing nonrepetitive sequences that have been used for genome editing. The repetitive sequences associated with TALEs can present some challenges, which the BuD array will not be subject to.

BuDN (Nuclease) and TALENs (from Stella et al. 2014)

BuDN (Nuclease) and TALENs (from Stella et al. 2014)

 

 

 

 

So, genome-editing tools are being developed at a remarkable pace. It will be important for those interested in Insect Genetic Technologies to remain aware of the emergence of new platforms since each will have its advantages and disadvantages relative to their intended application.

Take a look at this paper for the interesting details.
BuD, a helix-loop-helix DNA-binding domain for genome modification
S. Stella, R. Molina, B. López-Méndez, A. Juillerat, C. Bertonati, F. Daboussi, R. Campos-Olivas, P. Duchateau and G. Montoya
Acta Cryst. (2014). D70, 2042-2052
doi:10.1107/S1399004714011183

 

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