The generation of genome-modified animals is a powerful approach to analyze gene functions. The CAS9/guide RNA (gRNA) system is expected to become widely used for the efficient generation of genome-modified animals, but detailed studies on optimum conditions and availability are limited. In the present study, we attempted to generate large-scale genome-modified mice with an optimized CAS9/gRNA system, and confirmed the transmission of these mutations to the next generations. A comparison of different types of gRNA indicated that the target loci of almost all pups were modified successfully by the use of long-type gRNAs with CAS9. We showed that this system has much higher mutation efficiency and much lower off-target effect compared to zinc-finger nuclease. We propose that most of these off-target effects can be avoided by the careful control of CAS9 mRNA concentration and that the genome-modification efficiency depends rather on the gRNA concentration. Under optimized conditions, large-scale (∼10 kb) genome-modified mice can be efficiently generated by modifying two loci on a single chromosome using two gRNAs at once in mouse zygotes. In addition, the normal transmission of these CAS9/gRNA-induced mutations to the next generation was confirmed. These results indicate that CAS9/gRNA system can become a highly effective tool for the generation of genome-modified animals.

Preswallow bolus formation usually occurs in the mouth for liquids and in the oropharynx for solid foods. We examined the effect of chewing on the relationship between bolus transport and swallow initiation. Fifteen healthy subjects were imaged with lateral projection videofluorography while eating liquids, solid foods, and a mixture of liquid and solid foods in upright and facedown postures. Videotapes were reviewed to measure the location of the leading edge of the barium at swallow initiation. Chewing and initial consistency each altered the relationship between food transport and swallow initiation. In particular, when chewing liquid (or consuming foods with both liquid and solid phases), a portion of the food commonly reached the hypopharynx well before swallow onset. This transport to the hypopharynx was highly dependent on gravity, but transport to the valleculae for chewed solid food was active, depending primarily on tongue-palate contact. Chewing appeared to reduce the effectiveness of the posterior tongue-palate seal, allowing oral contents to spill into the pharynx. Consuming two-phase foods with both solid and liquid phases may increase the risk of aspiration in dysphagic individuals with impaired airway protective reflexes.

Duchenne muscular dystrophy (DMD) is an X-linked lethal muscle disorder caused by mutations in the Dmd gene encoding Dystrophin12. DMD model animals, such as mdx mice and canine X-linked muscular dystrophy dogs, have been widely utilized in the development of a treatment for DMD3. Here, we demonstrate the generation of Dmd-mutated rats using a clustered interspaced short palindromic repeats (CRISPR)/Cas system, an RNA-based genome engineering technique that is also adaptive to rats. We simultaneously targeted two exons in the rat Dmd gene, which resulted in the absence of Dystrophin expression in the F0 generation. Dmd-mutated rats exhibited a decline in muscle strength, and the emergence of degenerative/regenerative phenotypes in the skeletal muscle, heart, and diaphragm. These mutations were heritable by the next generation, and F1 male rats exhibited similar phenotypes in their skeletal muscles. These model rats should prove to be useful for developing therapeutic methods to treat DMD.

We developed a functional and versatile genome editing method using the CRISPR/Cas9 system in A. oryzae. This technique will contribute to the use of efficient targeted mutagenesis in many A. oryzae industrial strains.

Filamentous fungi are used for food fermentation and industrial production of recombinant proteins. They also serve as a source of secondary metabolites and are recently expected as hosts for heterologous production of useful secondary metabolites. Multiple-step genetic engineering is required to enhance industrial production involving these fungi, but traditional sequential modification of multiple genes using a limited number of selection markers is laborious. Moreover, efficient genetic engineering techniques for industrial strains have not yet been established. We have previously developed a clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9-based mutagenesis technique for the industrial filamentous fungus Aspergillus oryzae, enabling mutation efficiency of 10 to 20%. Here, we improved the CRISPR/Cas9 approach by including an AMA1-based autonomously replicating plasmid harboring the drug resistance marker ptrA. By using the improved mutagenesis technique, we successfully modified A. oryzae wild and industrial strains, with a mutation efficiency of 50 to 100%. Conditional expression of the Aoace2 gene from the AMA1-based plasmid severely inhibited fungal growth. This enabled forced recycling of the plasmid, allowing repeated genome editing. Further, double mutant strains were successfully obtained with high efficiency by expressing two guide RNA molecules from the genome-editing plasmid. Cotransformation of fungal cells with the genome-editing plasmid together with a circular donor DNA enabled marker-free multiplex gene deletion/integration in A. oryzae. The presented repeatable marker-free genetic engineering approach for mutagenesis and gene deletion/integration will allow for efficient modification of multiple genes in industrial fungal strains, increasing their applicability. IMPORTANCE Multiple gene modifications of specific fungal strains are required for achieving industrial-scale production of enzymes and secondary metabolites. In the present study, we developed an efficient multiple genetic engineering technique for the filamentous fungus Aspergillus oryzae. The approach is based on a clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 system and recycling of an AMA1-based autonomous replicating plasmid. Because the plasmid harbors a drug resistance marker (ptrA), the approach does not require the construction of auxotrophic industrial strains prior to genome editing and allows for forced recycling of the gene-editing plasmid. The established plasmid-recycling technique involves an Aoace2-conditional expression cassette, whose induction severely impairs fungal growth. We used the developed genetic engineering techniques for highly efficient marker-free multiple gene deletion/integration in A. oryzae. The genome-editing approaches established in the present study, which enable unlimited repeatable genetic engineering, will facilitate multiple gene modification of industrially important fungal strains.

No abstract

The CRISPR/Cas9 system has been used for spatio-temporal gene modification through the ubiquitous expression of gRNA by an RNA polymerase III promoter and the controlled expression of Cas9 using a tissue-specific or inducible promoter. However, unexpected gene disruptions indicate the necessity of a tissue-specific or inducible expression of not only Cas9 but also gRNA. In the present study, we attempted to develop a CRISPR/Cas9 system that could express functional gRNAs and Cas9 by a single RNA polymerase II promoter and induce multi-loci disruptions in specific cells. To this end, we designed vectors expressing ribozyme-flanked gRNAs (RGRs) and Cas9 mRNAs simultaneously. We showed that the mono-promoter-driven vector induces gene disruptions at the target loci in HEK 293 cells after transfection. In addition, two target loci were disrupted simultaneously by the transfection of a mono-promoter-driven vector expressing two RGRs and Cas9 mRNA. Finally, we constructed a universal vector for use in the construction of plasmids to be applied to the present mono-promoter-driven CRISPR/Cas9 system. We have thus provided a versatile tool for generating gene disruptions by the CRISPR/Cas9 system; this system should contribute to a wide range of investigations, including studies on spatio-temporal gene functions.

The nucleotide sequences offic-1 involved in the cell filamentation induced by cyclic AMP (5), and the number of the cell division genes is increasing, but their interaction is not still clear because of the complexity of the interaction. We have focused on cyclic AMP (cAMP) as a possible regulatory factor in cell division. Although cAMP is not essential for growth in Escherichia coli because the cya mutant is viable, much evidence supports the idea that cAMP controls cell growth or cell division in E. coli (3,9,22,23,26). We isolated cAMP-requiring mutants with double mutations of cya and cid (24, 25). We have found that an increase in intracellular cAMP levels induced cell filamentation in afic-i mutant at high temperature (14,23,27). cAMP is required for anucleate cell production (9). The filamentation of ftsZ(Ts) is repressed in the cya or crp mutant (26). Interestingly, Hughes et al. showed that cAMP binds DnaA protein and has a regulatory role in DNA replication (8).It is important to know the structure of the fic gene to understand its function in relation to cAMP. In this study we sequenced the fic and fic-i genes and found that thefic gene has some sequence-similar regions to other cell division genes.Plasmid pHB3 (1, 13), containing the fic gene; pMK108 (13), containing thefic-i gene; and other subcloned plasmids used as the source of DNA for sequencing were purified by the procedure of Birnboim and Doly (2). For preparation of plasmids on a small scale, the alkaline sodium dodecyl sulfate method of Davis et al. (4, 16) was used. Restriction fragments digested by AluI, EcoRV, HaeIII, HincII, HpaII, RsaI, Sau3A, TaqI, and ThaI were subcloned into M13 vector mpl8 or mpl9 (28). We sequenced a 2.5-kilobase-pair (kb) AvaI-AvaI region which includes the fic-i gene and its surroundings, as reported previously (13). The sequence strategy is shown in Fig. 1. Almost all double-stranded DNAs were sequenced by the dideoxy-chain termination method (20) (shown by arrows in Fig. 1). The Maxam and Gilbert method (17) was used for the parts (dashed arrows in * Corresponding author. Fig. 1) which are difficult to analyze by the M13 chain termination method. In the base sequence (Fig. 2) Fig. 2. The calculated protein size from the sequence of fic-i was 22,930 daltons, coinciding well with the protein product of 21 kilodaltons identified by the maxicell method, as reported previously (13).To find where in the DNA the mutation was generated in thefic-i gene, we sequenced, by the dideoxy-chain termination method, a 1.3-kb MluI-EcoRV region containing thefic gene. The fic DNA was isolated from pHB3. An enzymatically digested DNA fragment was inserted into an mpl8 or mpl9 vector, and sequencing was performed (detailed sequence strategy is not shown). G at position 1574 was changed to C in the fic-i mutant (Fig. 2). By this mutation,