Efficient disruption of endogenous Bombyx gene by TAL effector nucleases

Engineered nucleases are proteins that are able to cleave DNA at specified sites in the genome. These proteins have recently been used for gene targeting in a number of organisms. We showed earlier that zinc finger nucleases (ZFNs) can be used for generating gene-specific mutations in Bombyx mori by an error-prone DNA repair process of non-homologous end joining (NHEJ). Here we test the utility of another type of chimeric nuclease based on bacterial TAL effector proteins in order to induce targeted mutations in silkworm DNA. We designed three TAL effector nucleases (TALENs) against the genomic locus BmBLOS2, previously targeted by ZFNs. All three TALENs were able to induce mutations in silkworm germline cells suggesting a higher success rate of this type of chimeric enzyme. The efficiency of two of the tested TALENs was slightly higher than of the successful ZFN used previously. Simple design, high frequency of candidate targeting sites and comparable efficiency of induction of NHEJ mutations make TALENs an important alternative to ZFNs.

Graphical abstractFigure optionsDownload full-size imageDownload high-quality image (69 K)Download as PowerPoint slideHighlights► We designed three TAL-effector nucleases against Bombyx genomic locus BmBLOS2. ► Our TALEN constructs encoded full-length N and C-termini of TAL proteins. ► We confirmed the activity of all three engineered nucleases in yeast assays. ► All three engineered nucleases induced desired mutations in Bombyx germline.