CRISPR Advancements for Human Health.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) has emerged as a powerful gene editing technology that is revolutionizing biomedical research and clinical medicine. The CRISPR system allows scientists to rewrite the genetic code in virtually any organism. This review provides a comprehensive overview of CRISPR and its clinical applications. We first introduce the CRISPR system and explain how it works as a gene editing tool. We then highlight current and potential clinical uses of CRISPR in areas such as genetic disorders, infectious diseases, cancer, and regenerative medicine. Challenges that need to be addressed for the successful translation of CRISPR to the clinic are also discussed. Overall, CRISPR holds great promise to advance precision medicine, but ongoing research is still required to optimize delivery, efficacy, and safety.

Development of Polarity-Reversed Endometrial Epithelial Organoids.

The uterine epithelium is composed of a single layer of hormone responsive polarized epithelial cells that line the lumen and form tubular glands. Endometrial epithelial organoids (EEO) can be generated from uterine epithelia and recapitulate cell composition and hormone responses in vitro. As such, the development of EEO represents a major advance for facilitating mechanistic studies in vitro. However, a major limitation for the use of EEO cultured in basement membrane extract and other hydrogels is the inner location of apical membrane, thereby hindering direct access to the apical surface of the epithelium to study interactions with the embryo or infectious agents such as viruses and bacteria. Here, a straightforward strategy was developed that successfully reverses the polarity of EEO. The result is an apical-out organoid that preserves a distinct apical-basolateral orientation and remains responsive to ovarian steroid hormones. Our investigations highlight the utility of polarity-reversed EEO to study interactions with E. coli and blastocysts. This method of generating apical-out EEO lays the foundation for developing new in vitro functional assays, particularly regarding epithelial interactions with embryos during pregnancy or other luminal constituents in a pathological or diseased state.

Development of Polarity-Reversed Endometrial Epithelial Organoids.

The uterine epithelium is composed of a single layer of hormone responsive polarized epithelial cells that line the lumen and form tubular glands. Endometrial epithelial organoids (EEO) can be generated from uterine epithelia and recapitulate cell composition and hormone responses in vitro . As such, the development of EEO represents a major advance for facilitating mechanistic studies in vitro . However, a major limitation for the use of EEO cultured in basement membrane extract and other hydrogels is the inner location of apical membrane, thereby hindering direct access to the apical surface of the epithelium to study interactions with the embryo or infectious agents such as viruses and bacteria. Here, a straightforward strategy was developed that successfully reverses the polarity of EEO. The result is an apical-out organoid that preserves a distinct apical-basolateral orientation and remains responsive to ovarian steroid hormones. Our investigations highlight the utility of polarity-reversed EEO to study interactions with E. coli and blastocysts. This method of generating apical-out EEO lays the foundation for developing new in vitro functional assays, particularly regarding epithelial interactions with embryos during pregnancy or other luminal constituents in a pathological or diseased state.

Establishment, characterization, and validation of novel porcine embryonic fibroblasts as a potential source for genetic modification.

Fibroblasts are the common cell type in the connective tissue-the most abundant tissue type in the body. Fibroblasts are widely used for cell culture, for the generation of induced pluripotent stem cells (iPSCs), and as nuclear donors for somatic cell nuclear transfer (SCNT). We report for the first time, the derivation of embryonic fibroblasts (EFs) from porcine embryonic outgrowths, which share similarities in morphology, culture characteristics, molecular markers, and transcriptional profile to fetal fibroblasts (FFs). We demonstrated the efficient use of EFs as nuclear donors in SCNT, for enhanced post-blastocyst development, implantation, and pregnancy outcomes. We further validated EFs as a source for CRISPR/Cas genome editing with overall editing frequencies comparable to that of FFs. Taken together, we established an alternative and efficient pipeline for genome editing and for the generation of genetically engineered animals.

Newcastle disease virus vectors expressing consensus sequence of the H7 HA protein protect broiler chickens and turkeys against highly pathogenic H7N8 virus.

Continuous outbreaks of highly pathogenic avian influenza (HPAI) viruses in commercial poultry have caused devastating losses to domestic poultry with a raising public health concern. The outbreaks of HPAI viruses have increased worldwide, including the North America. Therefore, vaccination has been considered as an alternative strategy for an efficient control of HPAI viruses. In this study, we aimed to generate Newcastle disease virus (NDV) vectored H7 serotype-specific vaccines by expressing the consensus sequence of the HA protein. Conventional NDV strain LaSota vector and a chimeric NDV vector containing the avian paramyxovirus type-2 F and HN protein were able to express the consensus sequence of HA protein. The protective efficacy of vaccines was evaluated in broiler chickens and in turkeys. One-day-old poults were prime immunized with the chimeric vector expressing the HA protein followed by boost immunization with LaSota vector expressing the HA protein or co-expressing the HA and NA proteins. Our vaccine candidates provided complete protection of broiler chickens from mortality and shedding of H7N8 HPAI challenge virus. Turkeys were better protected by boosting with the LaSota vector co-expressing the HA and NA proteins than the LaSota vector expressing only the HA protein. Our study demonstrated a potential use of heterologous prime and boost vaccination strategy to protect poultry against H7 HPAI viruses.

Pathogenicity and Transmissibility of North American H7 Low Pathogenic Avian Influenza Viruses in Chickens and Turkeys.

Low pathogenic avian influenza (LPAI) viruses can silently circulate in poultry and wild aquatic birds and potentially mutate into highly pathogenic avian influenza (HPAI) viruses. In the U.S., recent emergence and spread of H7N8 and H7N9 HPAI viruses not only caused devastating losses to domestic poultry but also underscored the capability of LPAI viruses to mutate into HPAI viruses. Therefore, in this study, we evaluated pathogenicity and transmissibility of H7N8 and H7N9 LPAI viruses (the progenitors of HPAI viruses) in chickens and turkeys. We also included H7N2 isolated from an outbreak of LPAI in commercial chickens. H7 viruses replicated more efficiently in the respiratory tract than in the gastrointestinal tract, suggesting that their replication is restricted to the upper respiratory tract. Specifically, H7N2 replicated most efficiently in two-week-old chickens and turkeys. In contrast, H7N8 replicated least efficiently in those birds. Further, replication of H7N2 and H7N9 was restricted in the upper respiratory tract of four-week-old specific-pathogen-free (SPF) and broiler chickens. Despite their restricted replication, the two viruses efficiently transmitted from infected to naïve birds by direct contact, leading to seroconversion of contacted chickens. Our findings suggest the importance of continuous monitoring and surveillance of LPAI viruses in the fields.