Gene activation by dCas9-CBP and the SAM system differ in target preference.

Gene overexpression through the targeting of transcription activation domains to regulatory DNA via catalytically defective Cas9 (dCas9) represents a powerful approach to investigate gene function as well as the mechanisms of gene control. To date, the most efficient dCas9-based activator is the Synergistic Activation Mediator (SAM) system whereby transcription activation domains are directly fused to dCas9 as well as tethered through MS2 loops engineered into the gRNA. Here, we show that dCas9 fused to the catalytic domain of the histone acetyltransferase CBP is a more potent activator than the SAM system at some loci, but less efficient at other locations in Drosophila cells.

Targeted mutagenesis and functional analysis of adipokinetic hormone-encoding gene in Drosophila.

Adipokinetic hormones (Akhs) are small peptides (8-10 amino acid [aa] residues long) found in insects that regulate metabolic responses to stress by stimulating catabolic reactions and mobilizing energy stores. We employed Transcription activator-like effector nuclease (TALEN) mutagenesis and isolated an Akh(1) mutant carrying a small deletion in the gene that resulted in a truncated peptide; the second aa (Leu) was missing from the functional octapeptide. This null Dmel/Akh mutant is suitable to study Akh function without any effect on the C-terminal associated peptide encoded by the same gene.

The use of TALENs for nonhomologous end joining mutagenesis in silkworm and fruitfly.

Transcription activator-like effector nucleases (TALENs) are custom-made enzymes designed to cut double-stranded DNA at desired locations. The DNA breaks are repaired either by error-prone non-homologous end-joining (NHEJ) pathway or via homologous recombination requiring homologous DNA as a template for the repair. TALENs are used for site-specific mutagenesis in an extended range of organisms including insects.

Efficient TALEN construction for Bombyx mori gene targeting.

Engineered nucleases are artificial enzymes able to introduce double stranded breaks at desired genomic locations. The double stranded breaks start the error-prone repair process of non-homologous end-joining (NHEJ), which eventually leads to the induction of mutations at target sites. We showed earlier that ZFNs and TALENs are able to induce NHEJ mutations in the B.

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).

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.