Hideki Sezutsu

Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9

Genome engineering using programmable nucleases enables homologous recombination (HR)-mediated gene knock-in. However, the labour used to construct targeting vectors containing homology arms and difficulties in inducing HR in some cell type and organisms represent technical hurdles for the application of HR-mediated knock-in technology. Here, we introduce an alternative strategy for gene knock-in using transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) mediated by microhomology-mediated end-joining, termed the PITCh (Precise Integration into Target Chromosome) system. TALEN-mediated PITCh, termed TAL-PITCh, enables efficient integration of exogenous donor DNA in human cells and animals, including silkworms and frogs. We further demonstrate that CRISPR/Cas9-mediated PITCh, termed CRIS-PITCh, can be applied in human cells without carrying the plasmid backbone sequence. Thus, our PITCh-ing strategies will be useful for a variety of applications, not only in cultured cells, but also in various organisms, including invertebrates and vertebrates.

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. mori genome. In order to optimize our mutagenesis protocol, we modified one of the reported truncated TALEN scaffolds and optimized it for use in the B. mori embryo. We also established a novel B. mori somatic cell assay suitable for the preselection of highly efficient TALENs directly in the B. mori model system. We compared the efficiency of several TALEN pairs based on three different frameworks using the BmBLOS2 gene. The new active TALENs show one order of magnitude higher efficiency than those we used previously. We confirmed the utility of our improved protocol by mutagenesis of the autosomal gene, red egg (Bm-re) and showed that it allows obtaining homozygous mutants in G1. Our procedure minimizes the chance of failure in B. mori gene targeting experiments.

Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle.

Significance The juvenile–adult transition is a key developmental process in organisms. A long-held paradigm in insect endocrinology is that juvenile hormones (JHs) prevent metamorphosis until larvae attain an appropriate size for the juvenile–adult transition. However, little is known about the roles for JHs during embryonic and very early larval stages. We established knockouts of the silkworm, a classic model insect, and show that embryogenesis and maintenance of juvenile status during the early larval stages are largely independent of JHs or the JH-signaling pathway. Our results also suggest that an unidentified factor or signal is required to acquire the competence for metamorphosis. The presence of this factor has long been overlooked because JHs may conceal its action. Insect juvenile hormones (JHs) prevent precocious metamorphosis and allow larvae to undergo multiple rounds of status quo molts. However, the roles of JHs during the embryonic and very early larval stages have not been fully understood. We generated and characterized knockout silkworms (Bombyx mori) with null mutations in JH biosynthesis or JH receptor genes using genome-editing tools. We found that embryonic growth and morphogenesis are largely independent of JHs in Bombyx and that, even in the absence of JHs or JH signaling, pupal characters are not formed in first- or second-instar larvae, and precocious metamorphosis is induced after the second instar at the earliest. We also show by mosaic analysis that a pupal specifier gene broad, which is dramatically up-regulated in the late stage of the last larval instar, is essential for pupal commitment in the epidermis. Importantly, the mRNA expression level of broad, which is thought to be repressed by JHs, remained at very low basal levels during the early larval instars of JH-deficient or JH signaling-deficient knockouts. Therefore, our study suggests that the long-accepted paradigm that JHs maintain the juvenile status throughout larval life should be revised because the larval status can be maintained by a JH-independent mechanism in very early larval instars. We propose that the lack of competence for metamorphosis during the early larval stages may result from the absence of an unidentified broad-inducing factor, i.e., a competence factor.

Phenotypic characterization of a new Grin1 mutant mouse generated by ENU mutagenesis

In the RIKEN large‐scale N‐ethyl‐N‐nitrosourea (ENU) mutagenesis project we screened mice with a dominant mutation that exhibited abnormal behavior in the open‐field test, passive avoidance test and home‐cage activity test. We tested 2045 progeny of C57BL/6J males treated with ENU and untreated DBA/2J females in the open‐field test and isolated behavioral mutant M100174, which exhibited a significant increase in spontaneous locomotor activity. We identified a missense mutation in the Grin1 gene, which encodes NMDA receptor subunit 1, and designated the mutant gene Grin1Rgsc174. This mutation results in an arginine to cysteine substitution in the C0 domain of the protein. Detailed analyses revealed that Grin1Rgsc174 heterozygote exhibited increased novelty‐seeking behavior and slight social isolation in comparison with the wild type. In contrast to other Grin1 mutant mice, this mutant exhibited no evidence of heightened anxiety. These results indicate that this is a unique behavioral Grin1 gene mutant mouse that differs from the known Grin1 mutant mice. The results of immunohistochemical and biochemical analyses suggested that impaired interaction between the glutamatergic pathway and dopaminergic pathway may underlie the behavioral phenotypes of the Grin1Rgsc174 mutant.

Targeted disruption of a single sex pheromone receptor gene completely abolishes in vivo pheromone response in the silkmoth

Male moths use species-specific sex pheromones to identify and orientate toward conspecific females. Odorant receptors (ORs) for sex pheromone substances have been identified as sex pheromone receptors in various moth species. However, direct in vivo evidence linking the functional role of these ORs with behavioural responses is lacking. In the silkmoth, Bombyx mori, female moths emit two sex pheromone components, bombykol and bombykal, but only bombykol elicits sexual behaviour in male moths. A sex pheromone receptor BmOR1 is specifically tuned to bombykol and is expressed in specialized olfactory receptor neurons (ORNs) in the pheromone sensitive long sensilla trichodea of male silkmoth antennae. Here, we show that disruption of the BmOR1 gene, mediated by transcription activator-like effector nucleases (TALENs), completely removes ORN sensitivity to bombykol and corresponding pheromone-source searching behaviour in male moths. Furthermore, transgenic rescue of BmOR1 restored normal behavioural responses to bombykol. Our results demonstrate that BmOR1 is required for the physiological and behavioural response to bombykol, demonstrating that it is the receptor that mediates sex pheromone responses in male silkmoths. This study provides the first direct evidence that a member of the sex pheromone receptor family in moth species mediates conspecific sex pheromone information for sexual behaviour.

Positional cloning of a Bombyx pink-eyed white egg locus reveals the major role of cardinal in ommochrome synthesis.

Ommochromes are major insect pigments involved in coloration of compound eyes, eggs, epidermis and wings. In the silkworm Bombyx mori, adult compound eyes and eggs contain a mixture of the ommochrome pigments such as ommin and xanthommatin. Here, we identified the gene involved in ommochrome biosynthesis by positional cloning of B. mori egg and eye color mutant pink-eyed white egg (pe). The recessive homozygote of pe has bright red eyes and white or pale pink eggs instead of a normal dark coloration due to the decrease of dark ommochrome pigments. By genetic linkage analysis, we narrowed down the pe-linked region to ~258 kb, containing 17 predicted genes. RNA sequencing analyses showed that the expression of one candidate gene, the ortholog of Drosophila haem peroxidase cardinal, coincided with egg pigmentation timing, similar to other ommochrome-related genes such as Bm-scarlet and Bm-re. In two pe strains, a common missense mutation was found within a conserved motif of B. mori cardinal homolog (Bm-cardinal). RNA interference-mediated knockdown and transcription activator-like effector nuclease (TALEN)-mediated knockout of the Bm-cardinal gene produced the same phenotype as pe in terms of egg, adult eye and larval epidermis coloration. A complementation test of the pe mutant with the TALEN-mediated Bm-cardinal-deficient strain showed that the mutant phenotype could not be rescued, indicating that Bm-cardinal is responsible for pe. Moreover, knockdown of the cardinal homolog in Tribolium castaneum also induced red compound eyes. Our results indicate that cardinal plays a major role in ommochrome synthesis of holometabolous insects.

Disruption of diapause induction by TALEN-based gene mutagenesis in relation to a unique neuropeptide signaling pathway in Bombyx

The insect neuropeptide family FXPRLa, which carries the Phe-Xaa-Pro-Arg-Leu-NH2 sequence at the C-terminus, is involved in many physiological processes. Although ligand–receptor interactions in FXPRLa signaling have been examined using in vitro assays, the correlation between these interactions and in vivo physiological function is unclear. Diapause in the silkworm, Bombyx mori, is thought to be elicited by diapause hormone (DH, an FXPRLa) signaling, which consists of interactions between DH and DH receptor (DHR). Here, we performed transcription activator-like effector nuclease (TALEN)-based mutagenesis of the Bombyx DH-PBAN and DHR genes and isolated the null mutants of these genes in a bivoltine strain. All mutant silkworms were fully viable and showed no abnormalities in the developmental timing of ecdysis or metamorphosis. However, female adults oviposited non-diapause eggs despite diapause-inducing temperature and photoperiod conditions. Therefore, we conclude that DH signaling is essential for diapause induction and consists of highly sensitive and specific interactions between DH and DHR selected during ligand–receptor coevolution in Bombyx mori.

phiC31-integrase-mediated, site-specific integration of transgenes in the silkworm, Bombyx mori (Lepidoptera: Bombycidae)

Transgenic silkworms can be useful for investigating the functions of genes in the post-genomic era. However, the common method of using a transposon as an insertion tool may result in the random integration of a foreign gene into the genome and suffer from a strong position effect. To overcome these problems, it is necessary to develop a site-specific integration system. It is known that phiC31 integrase has the capacity to mediate recombination between the target sequences attP and attB. To test the availability of site-specific integration in the silkworm, we first examined the efficiency of recombination between the target sites of the two plasmids in silkworm embryos and found that the frequency of recombination was very high. Then we constructed a host strain that possessed the target sequence attP using the common method. We injected the donor plasmid together with the phiC31 integrase mRNA into the embryos of the host strain and obtained positive lines. Structural analysis of the lines showed that site-specific integration occurred by recombination between the genomic attP site and the attB site of the donor plasmid. We can conclude from the results that phiC31 integrase has the ability to mediate the site-specific integration of transgenes into the silkworm chromosome.

Identification of a Novel Strong and Ubiquitous Promoter/Enhancer in the Silkworm Bombyx mori

Transgenic techniques offer a valuable tool for determining gene functions. Although various promoters are available for use in gene overexpression, gene knockdown, and identification of transgenic individuals, there is nevertheless a lack of versatile promoters for such studies, and this dearth acts as a bottleneck, especially with regard to nonmodel organisms. Here, we succeeded in identifying a novel strong and ubiquitous promoter/enhancer in the silkworm. We identified a unique silkworm strain whose reporter gene showed strong and ubiquitous expression during the establishment of enhancer trap strains. In this strain, the transposon was inserted into the 5′UTR of hsp90, a housekeeping gene that is abundantly expressed in a range of tissues. To determine whether the promoter/enhancer of hsp90 could be used to induce strong gene expression, a 2.9-kb upstream genomic fragment of hsp90 was isolated (hsp90P2.9k), and its transcriptional activation activity was examined. Strikingly, hsp90P2.9k induced strong gene expression in silkworm cell cultures and also strongly induced gene expression in various tissues and developmental stages of the silkworm. hsp90P2.9k also exhibited significant promoter/enhancer activity in Sf9, a cell culture from the armyworm, suggesting that this fragment might possibly be used as a gene expression tool in other Lepidoptera. We further found that 2.0 kb of hsp90P2.9k is sufficient for the induction of strong gene expression. We believe that this element will be of value for a range of studies such as targeted gene overexpression, gene knockdown and marker gene expression, not only in the silkworm but also in other insect species.

Precise genome editing in the silkworm Bombyx mori using TALENs and ds- and ssDNA donors - A practical approach.

Engineered nucleases are able to introduce double stranded breaks at desired genomic locations. The breaks can be repaired by an error-prone non-homologous end joining (NHEJ) mechanism, or the repair process can be exploited to introduce precise DNA modifications by homology-directed repair (HDR) when provided with a suitable donor template. We designed a series of DNA donors including long dsDNA plasmids as well as short ssDNA oligonucleotides and compared the effectiveness of their utilization during gene targeting with highly efficient transcription activator-like effector nucleases (TALENs). While the use of long dsDNA donors for the incorporation of larger DNA fragments in Bombyx is still a problem, short single-stranded oligodeoxynucleotides (ssODNs) are incorporated quite efficiently. We show that appropriately designed ssODNs were integrated into germ cells in up to 79% of microinjected individuals and describe in more detail the conditions for the precise genome editing of Bombyx genes. We specify the donor sequence requirements that affected knock-in efficiency, and demonstrate the successful applications of this method of sequence deletion, insertion and replacement in the Bombyx genome.