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![[Figure 4]](cb5061fig4mag.jpg)
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Figure 4
Engineered BuDNs can target a DNA sequence in a cellular scenario. (a) Nuclease activity of BuDN towards its homodimeric target in yeast. Upon mating, the BuDNs generate a double-strand break at the site of interest, allowing the restoration of a functional lacZ gene by single-strand annealing (SSA), enabling the generation of a blue colour in the presence of X-Gal. The colour was quantified and scored as an Afilter value, a parameter correlated to the nuclease activity. (b) Sketch of the BuDN design (see Supporting Information). A BuD array (cyan) targeting the desired DNA sequence was fused to FokI similarly to an AvrBs3-based TALEN (purple). (c) A pair of BuDNs targeting the AvrBs3 sequence (Bs3) was built to compare its activity with AvrBs3-based TALEN. The different DNA targets used in the assay are shown. The Bs3 DNA contains two identical Bs3 binding sites in opposite orientations separated by a 15 bp DNA spacer. Bs3 A11′G C17′T and C15′A T18′C DNAs contain two base-pair substitutions each in only one of the Bs3 binding sites. (d) Nuclease activity of the BuDNs and TALEN towards the DNA targets. The grey dashed line indicates the experimental background level. (e) Comparison of the nuclease activity of both scaffolds towards the same target at different temperatures. BuDNs are sensitive to variations in the target sequence, while TALEN seem to ignore the mutations in the DNA. The background level has been subtracted from the histograms. The obtained values are an average of three independent experiments. See Supporting Information for a detailed description of the nucleases. |
![[Figure 4]](cb5061fig4.jpg)
 | STRUCTURAL BIOLOGY |
ISSN: 2059-7983
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