Cytosine base editing systems with minimized off-target effect and molecular size.

Cytosine base editing enables the installation of specific point mutations without double-strand breaks in DNA and is advantageous for various applications such as gene therapy, but further reduction of off-target risk and development of efficient delivery methods are desired. Here we show structure-based rational engineering of the cytosine base editing system Target-AID to minimize its off-target effect and molecular size. By intensive and careful truncation, DNA-binding domain of its deaminase PmCDA1 is eliminated and additional mutations are introduced to restore enzyme function.

Deaminase-mediated multiplex genome editing in Escherichia coli.

In eukaryotes, the CRISPR-Cas9 system has now been widely used as a revolutionary genome engineering tool. However, in prokaryotes, the use of nuclease-mediated genome editing tools has been limited to negative selection for the already modified cells because of its lethality. Here, we report on deaminase-mediated targeted nucleotide editing (Target-AID) adopted in Escherichia coli.

Beyond Native Cas9: Manipulating Genomic Information and Function.

Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated manipulation of genomic information is becoming more versatile by combining nuclease-deficient CRISPR systems with a wide variety of effectors including base-editing deaminases, transcriptional regulators, and epigenetic modifiers. The programmable binding ability of CRISPR systems is essential when the systems are employed as targeting domains to recruit the effectors to specific genomic loci. The discovery of a variety of Cas9 orthologs and engineered variants enables high-fidelity genome editing and a wider selection of genomic targets, and CRISPR-mediated deaminases enable more precise and predictable genome editing compared with CRISPR nuclease-based editing.

Deep-sea vent phage DNA polymerase specifically initiates DNA synthesis in the absence of primers.

A DNA polymerase is encoded by the deep-sea vent phage NrS-1. NrS-1 has a unique genome organization containing genes that are predicted to encode a helicase and a single-stranded DNA (ssDNA)-binding protein. The gene for an unknown protein shares weak homology with the bifunctional primase-polymerases (prim-pols) from archaeal plasmids but is missing the zinc-binding domain typically found in primases.

Dynamics of polycomb proteins-mediated histone modifications during UV irradiation-induced DNA damage.

Polycomb group (PcG) complexes are known to be chromatin modifiers and transcriptional repressors. In this work, we reported that the histone-modifying PcG complexes are able to participate in the repair process of ultraviolet (UV)-induced DNA lesions in the silkworm, Bombyx mori. The silkworm cells with depletion of PcG genes showed hypersensitive to UV-C irradiation and increased inhibition of cell proliferation.

Flap endonuclease of bacteriophage T7: Possible roles in RNA primer removal, recombination and host DNA breakdown.

Gene 6 protein of bacteriophage T7 has 5'-3'-exonuclease activity specific for duplex DNA. We have found that gene 6 protein also has flap endonuclease activity. The flap endonuclease activity is considerably weaker than the exonuclease activity.

Roles of Piwi proteins in transcriptional regulation mediated by HP1s in cultured silkworm cells.

Piwi proteins are part of a superfamily of Argonaute proteins, which are one of the core components of the RNA silencing pathway in many eukaryotes. Piwi proteins are thought to repress the transposon expression both transcriptionally and post-transcriptionally. Recently, Drosophila melanogaster Piwi was recently reported to associate with chromatin and to interact directly with the Heterochromatin Protein 1 (HP1a).

Establishment of a Bombyx mori nucleopolyhedrovirus (BmNPV) hyper-sensitive cell line from the silkworm e21 strain.

Baculoviral expression systems, including those of Autographa californica multiple nucleopolyhedrovirus Bombyx mori nucleopolyhedrovirus (BmNPV), are used for recombinant protein production. Four B. mori-derived (BmN4, Bm5, Bmc140, and Bme21) cell lines were infected with recombinant BmNPV viruses expressing firefly luciferase or EGFP as reporters under the control of a viral polyhedrin promoter.

Post-translational modifications of the N-terminal tail of histone H3 in holocentric chromosomes of Bombyx mori.

Epigenetic information is encoded in post-translational modifications (PTMs) of histones. Various combinations of these marks contribute to the regulation of chromatin-templated DNA metabolisms. The histone code is gradually translated into biological responses in model organisms.

Efficient soluble protein production on transgenic silkworms expressing cytoplasmic chaperones.

Baculovirus expression systems (BES) are widely used for recombinant protein production in lepidopteran cells or larvae. However, even in BES, the insolubility of recombinant proteins sometimes makes their expression difficult. In this study, to improve the solubility and yield of foreign proteins, we constructed transgenic silkworms using silkworm heat-shock proteins, Hsp70 and Hsp40, or Hsc70 and Hsp90 co-chaperone Hop.

Molecular characterization, localization, and distribution of innexins in the silkworm, Bombyx mori.

Gap junctions that allow for a direct exchange of second messenger and ions are the most conserved cellular structures in multicellular organisms. We have isolated and characterized a Bombyx mori gene innexin3 that encodes a new member of the innexin family required for the early embryonic development. The BmINX3 mRNA was 1,814 nucleotide residues in length, and the deduced amino acid sequence of BmInx3 shared 74% similarity with Apis melifera innexin3.

Construction of gene expression systems in insect cell lines using promoters from the silkworm, Bombyx mori.

The promoter regions of the Bombyx mori HSC70-4 and B. mori TCTP genes characterized previously were used for the construction of a series of constitutive gene expression systems active in cultured cells. The relative abilities of these promoters were evaluated by comparing those of a silkworm actin A3 (BmActin3) promoter, which is used widely as the first choice.