ABSTRACT
Interspecies blastocyst complementation (IBC) provides a unique platform to study development and holds the potential to overcome worldwide organ shortages. Despite recent successes, brain tissue has not been achieved through IBC. Here, we developed an optimized IBC strategy based on C-CRISPR, which facilitated rapid screening of candidate genes and identified that Hesx1 deficiency supported the generation of rat forebrain tissue in mice via IBC. Xenogeneic rat forebrain tissues in adult mice were structurally and functionally intact. Cross-species comparative analyses revealed that rat forebrain tissues developed at the same pace as the mouse host but maintained rat-like transcriptome profiles. The chimeric rate of rat cells gradually decreased as development progressed, suggesting xenogeneic barriers during mid-to-late pre-natal development. Interspecies forebrain complementation opens the door for studying evolutionarily conserved and divergent mechanisms underlying brain development and cognitive function. The C-CRISPR-based IBC strategy holds great potential to broaden the study and application of interspecies organogenesis.
Subject(s)
Prosencephalon , Animals , Prosencephalon/metabolism , Prosencephalon/embryology , Mice , Rats , Blastocyst/metabolism , Female , CRISPR-Cas Systems/genetics , Transcriptome , Organogenesis , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Male , Mice, Inbred C57BLABSTRACT
Adenine nucleotide translocator 4 (Ant4), an ATP/ADP transporter expressed in the early phases of spermatogenesis, plays a crucial role in male fertility. While Ant4 loss causes early arrest of meiosis and increased apoptosis of spermatogenic cells in male mice, its other potential functions in male fertility remain unexplored. Here, we utilized Ant4 knockout mice to delineate the effects of Ant4-deficiency on male reproduction. Our observations demonstrated that Ant4-deficiency led to infertility and impaired testicular development, which was further investigated by evaluating testicular oxidative stress, autophagy, and inflammation. Specifically, the loss of Ant4 led to an imbalance of oxidation and antioxidants. Significant ultrastructural alterations were identified in the testicular tissues of Ant4-deficient mice, including swelling of mitochondria, loss of cristae, and accumulation of autophagosomes. Our results also showed that autophagic flux and AKT-AMPK-mTOR signaling pathway were affected in Ant4-deficient mice. Moreover, Ant4 loss increased the expression of pro-inflammatory factors. Overall, our findings underscored the importance of Ant4 in regulating oxidative stress, autophagy, and inflammation in testicular tissues. Taken together, these insights provided a nuanced understanding of the significance of Ant4 in testicular development.
Subject(s)
Autophagy , Mice, Knockout , Mitochondrial ADP, ATP Translocases , Oxidative Stress , Testis , Animals , Male , Testis/metabolism , Oxidative Stress/physiology , Mitochondrial ADP, ATP Translocases/metabolism , Mitochondrial ADP, ATP Translocases/genetics , Mice , Autophagy/physiology , Infertility, Male/metabolism , Spermatogenesis/physiology , Apoptosis/physiology , Signal Transduction/physiologyABSTRACT
PURPOSE: Multiple factors, such as dietary patterns, pharmaceutical interventions, and exposure to harmful substances, possess the capacity to influence gut microbiota composition. Gut microbiota dysbiosis has emerged as a significant contributor to the progression of chronic kidney disease (CKD) and its associated complications. By comprehending the intricacies of the intestinal microbiota, this research endeavor holds the potential to offer novel perspectives on potential strategies for mitigating CKD progression. METHODS: In this retrospective analysis, we assessed gut microbiota composition in CKD patients. Fecal samples were collected from a cohort of 44 patients with stage 3-4 CKD, alongside a control group consisting of 132 healthy volunteers. Subsequently, 16 s rDNA sequencing was conducted to examine the composition of the gut microbiota. RESULTS: Our findings revealed significant alterations in the diversity of intestinal microbiota in fecal samples between patients with stage 3-4 CKD and healthy subjects. Among the 475 bacterial genera, 164 were shared, while 242 dominant genera were exclusive to healthy subjects and 69 to CKD stages 3-4 samples. Notably, healthy volunteers exhibited a prevalence of intestinal Firmicutes and Bacteroidetes, whereas stage 3-4 CKD patients displayed higher abundance of Proteobacteria and Actinobacteria. The presence of uncultured Coprobacillus sp. notably contributed to distinguishing between the two groups. ROC curve analysis identified distinct microbiota with superior diagnostic efficacy for discriminating stage 3-4 CKD patients from healthy individuals. Metabolic pathway analysis revealed differing dominant pathways between the two groups-the NADH dehydrogenase pathway in healthy individuals and the phosphate acetyltransferase pathway in stage 3-4 CKD patients. Moreover, the CKD cohort displayed a higher proportion of Gram-negative bacteria and facultative anaerobes. CONCLUSIONS: In conclusion, our study underscores the profound influence of gut microbiota dysbiosis on CKD progression. The distinct microbial profiles observed in CKD patients highlight the potential efficacy of microbiota-based interventions in mitigating CKD advancement.
Subject(s)
Gastrointestinal Microbiome , Renal Insufficiency, Chronic , Humans , Retrospective Studies , Dysbiosis/complications , Dysbiosis/microbiology , Renal Insufficiency, Chronic/metabolism , Feces/microbiology , BacteriaABSTRACT
The FokI catalytic domain can be fused to various DNA binding architectures to improve the precision of genome editing tools. However, evaluation of off-target effects is essential for developing these tools. We use Genome-wide Off-target analysis by Two-cell embryo Injection (GOTI) to detect low-frequency off-target editing events in mouse embryos injected with FokI-based architectures. Specifically, we test FokI-heterodimers fused with TALENs, FokI homodimers fused with RYdCas9, or FokI catalytic domains alone resulting in no significant off-target effects. These FokI genome editing systems exhibit undetectable off-target effects in mouse embryos, supporting the further development of these systems for clinical applications.
Subject(s)
Gene Editing , Genome , Animals , Mice , Catalytic Domain , Gene Editing/methods , CRISPR-Cas SystemsABSTRACT
CRISPR-Cas13 systems have recently been used for targeted RNA degradation in various organisms. However, collateral degradation of bystander RNAs has limited their in vivo applications. Here, we design a dual-fluorescence reporter system for detecting collateral effects and screening Cas13 variants in mammalian cells. Among over 200 engineered variants, several Cas13 variants including Cas13d and Cas13X exhibit efficient on-target activity but markedly reduced collateral activity. Furthermore, transcriptome-wide off-targets and cell growth arrest induced by Cas13 are absent for these variants. High-fidelity Cas13 variants show similar RNA knockdown activity to wild-type Cas13 but no detectable collateral damage in transgenic mice or adeno-associated-virus-mediated somatic cell targeting. Thus, high-fidelity Cas13 variants with minimal collateral effects are now available for targeted degradation of RNAs in basic research and therapeutic applications.
Subject(s)
CRISPR-Cas Systems , RNA , Animals , Mice , CRISPR-Cas Systems/genetics , RNA/genetics , RNA Stability/genetics , Mice, Transgenic , Transcriptome , Mammals/geneticsABSTRACT
Adenine nucleotide translocator 4 (Ant4), an ATP/ADP transporter expressed in the early phases of spermatogenesis, plays a crucial role in male fertility. While Ant4 loss causes early arrest of meiosis and increased apoptosis of spermatogenic cells in male mice, its other potential functions in male fertility remain unexplored. Here, we utilized Ant4 knockout mice to delineate the effects of Ant4-deficiency on male reproduction. Our observations demonstrated that Ant4-deficiency led to infertility and impaired testicular development, which was further investigated by evaluating testicular oxidative stress, autophagy, and inflammation. Specifically, the loss of Ant4 led to an imbalance of oxidation and antioxidants. Significant ultrastructural alterations were identified in the testicular tissues of Ant4-deficient mice, including swelling of mitochondria, loss of cristae, and accumulation of autophagosomes. Our results also showed that autophagic flux and AKT-AMPK-mTOR signaling pathway were affected in Ant4-deficient mice. Moreover, Ant4 loss increased the expression of pro-inflammatory factors. Overall, our findings underscored the importance of Ant4 in regulating oxidative stress, autophagy, and inflammation in testicular tissues. Taken together, these insights provided a nuanced understanding of the significance of Ant4 in testicular development.