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. The resulting tCDA1EQ is effective in N-terminal fusion (AID-2S) or inlaid architecture (AID-3S) with Cas9, showing minimized RNA-mediated editing and gRNA-dependent/independent DNA off-targets, as assessed in human cells. Combining with the smaller Cas9 ortholog system (SaCas9), a cytosine base editing system is created that is within the size limit of AAV vector.

Mammalian synthetic biology by CRISPRs engineering and applications.

Technologies harnessing CRISPR systems have been rapidly evolving and expanding the capacity of researchers for understanding of mammalian cell behavior and its underlying mechanisms through genome and epigenome manipulations. In this review, we summarized the recent developments of CRISPR-based technologies for genetic and epigenetic modifications that include engineering of Cas9 for PAM simplification, non-cleaving base editing tools and alteration of gene expression. Applications such as genome-wide screening methods or CRISPR-based DNA barcoding for cellular lineage tracking are highlighted. Anticipated and upcoming development for mammalian synthetic biology that includes organelle engineering is also discussed.

Deaminase-mediated multiplex genome editing in Escherichia coli.

In eukaryotes, the CRISPR-Cas9 system has now been widely used as a revolutionary genome engineering tool1, 2. 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 lethality3, 4. Here, we report on deaminase-mediated targeted nucleotide editing (Target-AID) 5 adopted in Escherichia coli. Cytidine deaminase PmCDA1 fused to the nuclease-deficient CRISPR-Cas9 system achieved specific point mutagenesis at the target sites in E. coli by introducing cytosine mutations without compromising cell growth. The cytosine-to-thymine substitutions were induced mainly within an approximately five-base window of target sequences on the protospacer adjacent motif-distal side, which can be shifted depending on the length of the single guide RNA sequence. Use of a uracil DNA glycosylase inhibitor 6 in combination with a degradation tag (LVA tag) 7 resulted in a robustly high mutation efficiency, which allowed simultaneous multiplex editing of six different genes. The major multi-copy transposase genes that consist of at least 41 loci were also simultaneously edited by using four target sequences. As this system does not rely on any additional or host-dependent factors, it may be readily applicable to a wide range of bacteria.

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. Finally, combining transcriptional regulators with CRISPR systems can control expression of specific genes in a genome. Some applications and future challenges of CRISPR-derived tools are also discussed.

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. We show that this gene product has efficient DNA polymerase activity and is processive in DNA synthesis in the presence of the NrS-1 helicase and ssDNA-binding protein. Remarkably, this NrS-1 DNA polymerase initiates DNA synthesis from a specific template DNA sequence in the absence of any primer. The de novo DNA polymerase activity resides in the N-terminal domain of the protein, whereas the C-terminal domain enhances DNA binding.

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. Interestingly, an SQ site in the silkworm-human chimeric H2A protein synthesized here was phosphorylated rapidly upon UV-C exposure, which could be used as a marker for monitoring the response to DNA damage in silkworm cells. Under these UV-C irradiated conditions, we found that PRC1-mediated ubiquitylation of H2AX, but not of H2AZ, were decreased and this deubiquitylation was independent of its phosphorylation event. In contrast, UV-C irradiation induced the increase of trimethylation of lysine 27 on histone H3 (H3K27me3), a mark of transcriptionally silent chromatin catalyzed by another PcG subcomplex, PRC2. Collectively, we provided the first evidence on chromatin remodeling in response to UV-C lesion in silkworm and revealed another layer role for PcG complexes-mediated histone modifications in contributing to creating an open chromatin structure for the efficient repair of DNA damages.

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. Unlike the human homolog of gene 6 protein, the flap endonuclease activity of gene 6 protein is dependent on the length of the 5'-flap. This dependency of activity on the length of the 5'-flap may result from the structured helical gateway region of gene 6 protein which differs from that of human flap endonuclease 1. The flap endonuclease activity provides a mechanism by which RNA-terminated Okazaki fragments, displaced by the lagging strand DNA polymerase, are processed. 3'-extensions generated during degradation of duplex DNA by the exonuclease activity of gene 6 protein are inhibitory to further degradation of the 5'-terminus by the exonuclease activity of gene 6 protein. The single-stranded DNA binding protein of T7 overcomes this inhibition.

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). However, similar interactions have not been reported in other higher eukaryotes. Here we show that silkworm Piwi proteins interact with HP1s in the nucleus. The silkworm, Bombyx mori, has two Piwi proteins, Ago3 and Siwi, and two typical HP1 proteins, HP1a and HP1b. We found that HP1a plays an important role in the interaction between Ago3/Siwi and HP1b in the ovary-derived BmN4 cell line. We also found that Ago3/Siwi regulates the transcription in an HP1-dependent manner. These results suggest that silkworm Piwi proteins function as a chromatin regulator in collaboration with HP1a and HP1b.

Flap endonuclease activity of gene 6 exonuclease of bacteriophage T7.

Flap endonucleases remove flap structures generated during DNA replication. Gene 6 protein of bacteriophage T7 is a 5'-3'-exonuclease specific for dsDNA. Here we show that gene 6 protein also possesses a structure-specific endonuclease activity similar to known flap endonucleases. The flap endonuclease activity is less active relative to its exonuclease activity. The major cleavage by the endonuclease activity occurs at a position one nucleotide into the duplex region adjacent to a dsDNA-ssDNA junction. The efficiency of cleavage of the flap decreases with increasing length of the 5'-overhang. A 3'-single-stranded tail arising from the same end of the duplex as the 5'-tail inhibits gene 6 protein flap endonuclease activity. The released flap is not degraded further, but the exonuclease activity then proceeds to hydrolyze the 5'-terminal strand of the duplex. T7 gene 2.5 single-stranded DNA-binding protein stimulates the exonuclease and also the endonuclease activity. This stimulation is attributed to a specific interaction between the two proteins because Escherichia coli single-stranded DNA binding protein does not produce this stimulatory effect. The ability of gene 6 protein to remove 5'-terminal overhangs as well as to remove nucleotides from the 5'-termini enables it to effectively process the 5'-termini of Okazaki fragments before they are ligated.

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. Bme21 exhibited significantly higher (100-fold) luciferase activity than BmN4 and Bm5. With the EGFP reporter protein, Bme21 cells showed a marked increase in the ratio of EGFP-positive cells, reaching 90 % on day 4 post-infection, while Bm5 and BmN4 cells had a slow increase in the ratio of their EGFP-positive population. The viral titer in a supernatant of Bme21 cell culture increased faster than those of Bm5 and BmN4 cells. This susceptibility indicates that the Bme21 cell line is useful for large-scale protein expression using BmNPV.

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. However, in the silkworm, the modifications of histones with unique holocentric chromosomes have not yet been analyzed. TAU-PAGE analysis of the silkworm histone variants H2A, H2B, and H3, separated by RP-HPLC, suggested silkworm specific modification. Detailed mass spectrometry analyses of the peptides derived from the N-terminus of the silkworm H3.2 generated by glutamyl endopeptidase, lysyl endopeptidase, and trypsin digestions revealed global modifications around H3K9.

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. In these transgenic silkworms, the expression levels of the transgenes were under the control of a UAS.hsp mini-promoter driven by a Gal4NFkBp65 activator. When the transgenic silkworm with HSP70 and 40 (TGS-HSP70/40) was infected with BmNPV carrying mC3d and Gal4NFkBp65 under the control of baculovirus polyhedrin or p10 promoters, respectively, the soluble fraction of the His- or His.GST-tagged mC3d increased significantly. Similarly, the transgenic silkworm with HSC70 and HOP (TGS-HOP7) was effective for the expression of a steroid hormone receptor, USP2. In conclusion, the His-tagged baculovirus expression system featuring the chaperone effect TGS-HSP70/40 and TGS-HOP7 silkworms is effective for increasing the yields of soluble and functional foreign gene products.

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. The expression profile of the BmINX3 mRNA is similar to that of previously described BmINX2, expressed in ovary and testis after 5th instar larvae and in fat body after gut purge. However, during embryogenesis, the expression of BmINX3 mRNA is restricted to the blastokinesis stage. Microscopic observation of the BmInx2 and BmInx3 fused to fluorescent proteins showed an overlapping cytoplasmic expression, whereas the BmInx4 is accumulated in the cytoplasmic surface at which two cells have physical contact. This finding of innexins distribution in silkworm would provide an essential basis for future studies of the functions and interactions of innexins.

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. A series of constitutive expression systems constructed were assayed for the transcription efficiency by connecting four reporter cDNAs, firefly luciferase, 3GFP, Ds-Red, and beta-galactosidase gene using the Gateway LR reaction. The insertion of an intron enhancer into the site between the TCTP promoter and gene increased the transcription of the BmTCTP promoter by 10-fold. The insertion of the IE-1 gene and HR3 enhancer to the all three promoters were found to increase the transcription up to 560 times.