Genome editing in mammalian cells using the CRISPR type I-D nuclease.

Adoption of CRISPR-Cas systems, such as CRISPR-Cas9 and CRISPR-Cas12a, has revolutionized genome engineering in recent years; however, application of genome editing with CRISPR type I-the most abundant CRISPR system in bacteria-remains less developed. Type I systems, such as type I-E, and I-F, comprise the CRISPR-associated complex for antiviral defense ('Cascade': Cas5, Cas6, Cas7, Cas8 and the small subunit) and Cas3, which degrades the target DNA; in contrast, for the sub-type CRISPR-Cas type I-D, which lacks a typical Cas3 nuclease in its CRISPR locus, the mechanism of target DNA degradation remains unknown. Here, we found that Cas10d is a functional nuclease in the type I-D system, performing the role played by Cas3 in other CRISPR-Cas type I systems. The type I-D system can be used for targeted mutagenesis of genomic DNA in human cells, directing both bi-directional long-range deletions and short insertions/deletions. Our findings suggest the CRISPR-Cas type I-D system as a unique effector pathway in CRISPR that can be repurposed for genome engineering in eukaryotic cells.

Real-world efficacy of proactive maintenance treatment with delgocitinib ointment twice weekly in adult patients with atopic dermatitis.

Previous studies have shown the efficacy of delgocitinib (DEL) ointment, a topical Janus kinase inhibitor, against atopic dermatitis (AD). However, there is no available information regarding the efficacy of DEL ointment in maintaining remission. Data of patients with AD who received remission maintenance therapy twice weekly with DEL or topical corticosteroid (TCS) on the affected skin of each upper limb were extracted from the medical records. Efficacy was assessed based on changes in pruritus numerical rating scale (NRS) score, stratum corneum hydration (SCH), erythema index (EI). Of 25 patients, four patients (16%) had eczema flare-ups on the TCS side and eight patients (32%) on the DEL side. The extent of change in each parameter between TCS- and DEL-treated areas of the skin did not differ significantly. The mean changes in the NRS and EI showed a slight improvement on the side treated with TCS and were slightly worse on the side treated with DEL. However, the SCH of the DEL group was maintained, while that of the TCS group worsened. TCS is more likely to be effective than DEL in terms of remission maintenance therapy. However, topical DEL is as effective as topical steroid in the maintenance therapy of AD in dry skin patients.

Selective DNA-binding of SP120 (rat ortholog of human hnRNP U) is mediated by arginine-glycine rich domain and modulated by RNA.

A human protein heterogeneous ribonucleoprotein U (hnRNP U) also known as Scaffold attachment factor A (SAF-A) and its orthologous rat protein SP120 are abundant and multifunctional nuclear protein that directly binds to both DNA and RNA. The C-terminal region of hnRNP U enriched with arginine and glycine is essential for the interaction with RNA and the N-terminal region of SAF-A termed SAP domain has been ascribed to the DNA binding. We have reported that rat hnRNP U specifically and cooperatively binds to AT-rich DNA called nuclear scaffold/matrix-associated region (S/MAR) although its detailed mechanism remained unclear. In the present study analysis of hnRNP U deletion mutants revealed for the first time that a C-terminal domain enriched with Arg-Gly (defined here as 'RG domain') is predominantly important for the S/MAR-selective DNA binding activities. RG domain alone directly bound to S/MAR and coexistence with the SAP domain exerted a synergistic effect. The binding was inhibited by netropsin, a minor groove binder with preference to AT pairs that are enriched in S/MAR, suggesting that RG domain interacts with minor groove of S/MAR DNA. Interestingly, excess amounts of RNA attenuated the RG domain-dependent S/MAR-binding of hnRNP U. Taken together, hnRNP U may be the key element for the RNA-regulated recognition of S/MAR DNA and thus contributing to the dynamic structural changes of chromatin compartments.

Clinical course of more than 10 years in a patient with extensive skin burns who received cultured epidermal autograft transplantation.

Cultured epidermal autografts (CEAs) are used to treat extensive burns, giant congenital melanocytic nevi, and epidermolysis bullosa, but information about the long-term clinical course after CEA transplantation is scarce. Here we report 10 years' progress of a 7-year-old Japanese girl who suffered from a scald burn injury affecting 80% of her total body surface area and was treated with CEA transplantation. The skin of a child with extensive burns treated with CEAs appeared soft and of a good texture, even after 10 years, and recovery of skin pigmentation and scar condition were better at sites with a combination of CEAs and autologous skin grafts than those with CEAs alone.

Genome editing in plants using CRISPR type I-D nuclease.

Genome editing in plants has advanced greatly by applying the clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas system, especially CRISPR-Cas9. However, CRISPR type I-the most abundant CRISPR system in bacteria-has not been exploited for plant genome modification. In type I CRISPR-Cas systems, e.g., type I-E, Cas3 nucleases degrade the target DNA in mammals. Here, we present a type I-D (TiD) CRISPR-Cas genome editing system in plants. TiD lacks the Cas3 nuclease domain; instead, Cas10d is the functional nuclease in vivo. TiD was active in targeted mutagenesis of tomato genomic DNA. The mutations generated by TiD differed from those of CRISPR/Cas9; both bi-directional long-range deletions and short indels mutations were detected in tomato cells. Furthermore, TiD can be used to efficiently generate bi-allelic mutant plants in the first generation. These findings indicate that TiD is a unique CRISPR system that can be used for genome engineering in plants.

Two-pass dual-energy CT imaging for simultaneous detection, characterization, and volume measurement of urinary stones with excretory-phase CT urography alone: a phantom study.

PURPOSE: To evaluate if two-pass dual-energy CT imaging--i.e., simultaneous three-material and two-material decomposition analysis--can depict and characterize urinary stones in various concentrations of iodine solution in vitro. MATERIALS AND METHODS: Twelve urinary stones were scanned with a dual-source CT scanner. First, each stone (in a saline-filled tube) underwent single- and dual-energy mode CT scans in order to measure the volume of the stone. Each stone was then placed in various concentrations of contrast medium and scanned in dual-energy mode to calculate its volume via three-material decomposition analysis. Two-pass dual-energy CT imaging analysis software for the Matlab environment, which was developed specifically to process simultaneous three-material and two-material decomposition, was applied to characterize and calculate the volume of each stone. RESULTS: Although the virtual non-contrast images from three-material decomposition analysis clearly visualized all of the stones in contrast medium with up to 80 mgI/mL, the volumes of the uric acid stones were overestimated. Two-pass dual-energy CT imaging was able to depict and characterize non-uric-acid stones in diluted contrast medium with up to 80 mgI/mL, whereas uric acid stones were correctly evaluated in diluted contrast medium with 40 mgI/mL or less. CONCLUSIONS: Two-pass dual-energy CT imaging is able to depict and characterize urinary stones in contrast medium.