Selected Grantee Publications
- Clear All
- 56 results found
- CRISPR
- Spectrometry
Profiling Development of Abdominal Organs in the Pig
Gabriel et al., Scientific Reports. 2022.
https://www.doi.org/10.1038/s41598-022-19960-5
The pig is a model system for studying human development and disease due to its similarities to human anatomy, physiology, size, and genome. Moreover, advances in CRISPR gene editing have made genetically engineered pigs a viable model for the study of human pathologies and congenital anomalies. However, a detailed atlas illustrating pig development is necessary for identifying and modeling developmental defects. Here, the authors describe normal development of the pig abdominal system (i.e., kidney, liver, pancreas, spleen, adrenal glands, bowel, gonads) and compare them with congenital defects that can arise in gene-edited SAP130 mutant pigs. This atlas and the methods described here can be used as tools for identifying developmental pathologies of the abdominal organs in the pig at different stages of development. Supported by ORIP (U42OD011140), NHLBI, NIAID, NIBIB, NICHD, and NINDS.
A Multidimensional Metabolomics Workflow to Image Biodistribution and Evaluate Pharmacodynamics in Adult Zebrafish
Jackstadt et al., Disease Models & Mechanisms. 2022.
https://www.doi.org/10.1242/dmm.049550
The evaluation of tissue distribution and pharmacodynamic properties of a drug is essential but often expensive in clinical research. The investigators developed a multidimensional metabolomics platform to evaluate drug activity that integrates mass spectrometry–based imaging, absolute drug quantitation, in vivo isotope tracing, and global metabolome analysis in zebrafish. They validated this platform by evaluating whole-body distribution of the anti-rheumatic agent hydroxychloroquine sulfate and its impact on the systemic metabolism of adult zebrafish. This work suggests that the multidimensional metabolomics platform is a cost-effective method for evaluating on- and off-target effects of drugs. Supported by ORIP (R24OD024624) and NIEHS.
Effects of Ex Vivo Blood Anticoagulation and Preanalytical Processing Time on the Proteome Content of Platelets
Yunga et al., Journal of Thrombosis and Haemostasis. 2022.
https://www.doi.org/10.1111/jth.15694
The investigators studied how various blood anticoagulation options and processing times affect platelet function and protein content ex vivo. Using platelet proteome quantification and triple quadrupole mass spectrometry, they found that anticoagulant-specific effects on platelet proteomes included increased complement system and decreased α-granule proteins in platelets from EDTA-anticoagulated blood. Heparinized blood had higher levels of histone and neutrophil-associated proteins, as well as formation of platelet–neutrophil extracellular trap interactions in whole blood ex vivo. The study indicates that different anticoagulants and preanalytical processing times affect platelet function and platelet protein content ex vivo, suggesting more rigorous phenotyping strategies for platelet omics studies. Supported by ORIP (S10OD012246), NHLBI, NCI and NEI.
AAV5 Delivery of CRISPR-Cas9 Supports Effective Genome Editing in Mouse Lung Airway
Liang et al., Molecular Therapy. 2022.
https://www.cell.com/molecular-therapy-family/molecular-therapy/fulltext/S1525-0016(21)00530-X
Genome editing in the lung has the potential to provide long-term expression of therapeutic protein to treat lung genetic diseases. The authors illustrated that AAV5 can efficiently deliver CRISPR-Cas9 to mouse lung airways and was the first to achieve ∼20% editing efficiency in those airways. Results were confirmed through independent experiments at two different institutes. This highly efficient dual AAV platform will facilitate the study of genome editing in the lung and other tissue types. Supported by ORIP (U42OD026645).
Deep Learning Is Widely Applicable to Phenotyping Embryonic Development and Disease
Naert et al., Development. 2021.
https://pubmed.ncbi.nlm.nih.gov/34739029/
Genome editing simplifies the generation of new animal models for congenital disorders. The authors illustrate how deep learning (U-Net) automates segmentation tasks in various imaging modalities. They demonstrate this approach in embryos with polycystic kidneys (pkd1 and pkd2) and craniofacial dysmorphia (six1). They provide a library of pre-trained networks and detailed instructions for applying deep learning to datasets and demonstrate the versatility, precision, and scalability of deep neural network phenotyping on embryonic disease models. Supported by ORIP (P40OD010997, R24OD030008), NICHD, NIDDK, and NIMH.
MIC-Drop: A Platform for Large-scale In Vivo CRISPR Screens
Parvez et al., Science. 2021.
https://pubmed.ncbi.nlm.nih.gov/34413171/
CRISPR screens in animals are challenging because generating, validating, and keeping track of large numbers of mutant animals is prohibitive. These authors introduce Multiplexed Intermixed CRISPR Droplets (MIC-Drop), a platform combining droplet microfluidics, single-needle en masse CRISPR ribonucleoprotein injections, and DNA barcoding to enable large-scale functional genetic screens in zebrafish. In one application, they showed that MIC-Drop could identify small-molecule targets. Furthermore, in a MIC-Drop screen of 188 poorly characterized genes, they discovered several genes important for cardiac development and function. With the potential to scale to thousands of genes, MIC-Drop enables genome-scale reverse genetic screens in model organisms. Supported by ORIP (R24OD017870), NIGMS, and NHLBI.
TGF-β1 Signaling Is Essential for Tissue Regeneration in the Xenopus Tadpole Tail
Nakamura et al., Biochemical and Biophysical Research Communications. 2021.
https://www.sciencedirect.com/science/article/pii/S0006291X21008731
Amphibians, such as Xenopus tropicalis, exhibit a remarkable capacity for tissue regeneration after traumatic injury. Nakamura et al. show that inhibition of TGF-β1 function prevents tail regeneration in Xenopus tropicalis tadpoles. CRISPR-mediated knock-out (KO) of tgfb1 retards tail regeneration; the phenotype of tgfb1 KO tadpoles can be rescued by injection of tgfb1 mRNA. Cell proliferation, critical for tissue regeneration, is downregulated in tgfb1 KO tadpoles; tgfb1 KO reduces the expression of phosphorylated Smad2/3 (pSmad2/3). These results show that TGF-β1 regulates cell proliferation through the activation of Smad2/3. They propose that TGF-β1 plays a critical role in TGF-β receptor-dependent tadpole tail regeneration in Xenopus. Supported by ORIP (P40OD010997, R24OD030008).
Phase Separation Drives Aberrant Chromatin Looping and Cancer Development
Ahn et al., Nature. 2021.
https://doi.org/10.1038/s41586-021-03662-5
How unstructured intrinsically disordered regions (IDRs) contribute to oncogenesis is elusive. Using an Orbitrap fusion tribrid mass spectrometer, investigators show that IDRs contained within NUP98–HOXA9, a homeodomain-containing transcription factor chimera recurrently detected in leukaemias, are essential for establishing liquid–liquid phase separation (LLPS) puncta of chimera and for inducing leukaemic transformation. LLPS of NUP98–HOXA9 not only promotes chromatin occupancy of chimera transcription factors, but also is required for the formation of a broad “super-enhancer”-like binding pattern typically seen at leukaemogenic genes, which potentiates transcriptional activation. An artificial HOX chimera, created by replacing the phenylalanine and glycine repeats of NUP98 with an unrelated LLPS-forming IDR of the FUS protein, had similar enhancing effects on the genome-wide binding and target gene activation of the chimera. This report describes a proof-of-principle example in which cancer acquires mutation to establish oncogenic transcription factor condensates via phase separation, which simultaneously enhances their genomic targeting and induces organization of aberrant three-dimensional chromatin structure during tumor transformation. Supported by ORIP (S10OD018445).
Nonhuman Primate Models for SARS-CoV-2 Research: Cryopreservation as a Means to Maintain Critical Models and Enhance the Genetic Diversity of Colonies
Arnegard and Hild et al., Lab Animal. 2021.
https://doi.org/10.1038/s41684-021-00792-1
This commentary, written by ORIP staff, addresses the need for improved cryopreservation methods and resources for nonhuman primate (NHP) gametes and embryos to safeguard newly developed NHP models and enhance the genetic diversity of NHP colonies without reliance on animal importations. Cryopreservation also plays critical roles in medical approaches to preserve the fertility of patients who must undergo potentially gonadotoxic treatments, as well as nascent genome editing efforts to develop new NHP models for human diseases. Given these diverse benefits to research progress, ORIP continues to fund the development of cryopreservation tools and approaches for NHPs and other animal models.
Identification of Basp1 as a Novel Angiogenesis-regulating Gene by Multi-Model System Studies
Khajavi et al., FASEB Journal. 2021.
https://pubmed.ncbi.nlm.nih.gov/33899275/
The authors previously used genetic diversity in inbred mouse strains to identify quantitative trait loci (QTLs) responsible for differences in angiogenic response. Employing a mouse genome-wide association study (GWAS) approach, the region on chromosome 15 containing Basp1 was identified as being significantly associated with angiogenesis in inbred strains. To investigate its role in vivo, they knocked out basp1 in transgenic kdrl:zsGreen zebrafish embryos using a widely adopted CRISPR-Cas9 system. They further showed that basp1 promotes angiogenesis by upregulating β-catenin gene and the Dll4/Notch1 signaling pathway. These results provide the first in vivo evidence to indicate the role of basp1 as an angiogenesis-regulating gene. Supported by ORIP (R24OD017870) and NEI.