Targeted mutagenesis in the silkworm Bombyx mori using zinc finger nuclease mRNA injection

Targeted mutagenesis is one of the key methods for functional gene analysis. A simplified variant of gene targeting uses direct microinjection of custom-designed Zinc Finger Nuclease (ZFN) mRNAs into Drosophila embryos. To evaluate the applicability of this method to gene targeting in another insect, we mutagenized the Bombyx mori epidermal color marker gene BmBLOS2, which controls the formation of uric acid granules in the larval epidermis. Our results revealed that ZFN mRNA injection is effective to induce somatic, as well as germline, mutations in a targeted gene by non-homologous end joining (NHEJ). The ZFN-induced NHEJ mutations lack end-filling and blunt ligation products, and include mainly 7 bp or longer deletions, as well as single nucleotide insertions. These observations suggest that the B. mori double-strand break repair system relies on microhomologies rather than on a canonical ligase IV-dependent mechanism. The frequency of germline mutants in G1 was sufficient to be used for gene targeting relying on a screen based solely on molecular methods.

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► The absence of a general gene-targeting system for systematic reverse genetic studies has been a significant limitation in silkworm research. The authors performed direct zinc finger nuclease mRNA injection to B. mori embryos and induced mutations in an epidermal color gene BmBLOS2. The results suggest that the B. mori double-strand break repair system relies on microhomologies rather than on a ligase IV-dependent mechanism typical for Drosophila. The observed frequency of germline mutations was sufficient for future use in gene targeting relying solely on molecular methods of screening.