Programmable RNA-Guided Large DNA Transgenesis by CRISPR/Cas9 and Site-Specific Integrase Bxb1.

The inability to insert large DNA constructs into the genome efficiently and precisely is a key challenge in genomic engineering. Random transgenesis, which is widely used, lacks precision, and comes with a slew of drawbacks. Lentiviral and adeno-associated viral methods are plagued by, respectively, DNA toxicity and a payload capacity of less than 5 kb. Homology-directed repair (HDR) techniques based on CRISPR-Cas9 can be effective, but only in the 1-5 kb range. In addition, long homology arms-DNA sequences that permit construct insertion-of lengths ranging from 0.5 to 5 kb are required by currently known HDR-based techniques. A potential new method that uses Cas9-guided transposases to insert DNA structures up to 10 kb in length works well in bacteria, but only in bacteria. Surmounting these roadblocks, a new toolkit has recently been developed that combines RNA-guided Cas9 and the site-specific integrase Bxb1 to integrate DNA constructs ranging in length from 5 to 43 kb into mouse zygotes with germline transmission and into human cells. This ground-breaking toolkit will give researchers a valuable resource for developing novel, urgently needed mouse and human induced pluripotent stem cell (hiPSC) models of cancer and other genetic diseases, as well as therapeutic gene integration and biopharmaceutical applications, such as the development of stable cell lines to produce therapeutic protein products

Efficient targeted transgenesis of large donor DNA into multiple mouse genetic backgrounds using bacteriophage Bxb1 integrase.

The development of mouse models of human disease and synthetic biology research by targeted transgenesis of large DNA constructs represent a significant genetic engineering hurdle. We developed an efficient, precise, single-copy integration of large transgenes directly into zygotes using multiple mouse genetic backgrounds. We used in vivo Bxb1 mediated recombinase-mediated cassette exchange (RMCE) with a transgene landing pad composed of dual heterologous Bxb1 attachment (att) sites in cis, within the Gt(ROSA)26Sor safe harbor locus. RMCE of donor was achieved by microinjection of vector DNA carrying cognate attachment sites flanking the donor transgene with Bxb1-integrase mRNA. This approach achieves perfect vector-free integration of donor constructs at efficiencies \u3e 40% with up to ~ 43 kb transgenes. Coupled with a nanopore-based Cas9-targeted sequencing (nCATS), complete verification of precise insertion sequence was achieved. As a proof-of-concept we describe the development of C57BL/6J and NSG Krt18-ACE2 models for SARS-CoV2 research with verified heterozygous N1 animals within ~ 4 months. Additionally, we created a series of mice with diverse backgrounds carrying a single att site including FVB/NJ, PWK/PhJ, NOD/ShiLtJ, CAST/EiJ and DBA/2J allowing for rapid transgene insertion. Combined, this system enables predictable, rapid development with simplified characterization of precisely targeted transgenic animals across multiple genetic backgrounds

ADAM17 is essential for ectodomain shedding of the EGF-receptor ligand amphiregulin.

The epidermal growth factor (EGF)-receptor ligand amphiregulin (AREG) is a potent growth factor implicated in proliferative skin diseases and in primary and metastatic epithelial cancers. AREG, synthesized as a propeptide, requires conversion to an active peptide by metalloproteases by a process known as ectodomain shedding. Although (ADAM17) a disintegrin and metalloprotease 17 is a key sheddase of AREG, ADAM8-, ADAM15-, and batimastat (broad metalloprotease inhibitor)-sensitive metalloproteases have also been implicated in AREG shedding. In the present study, using a curly bare (Rhbdf2cub ) mouse model that shows loss-of-hair, enlarged sebaceous gland, and rapid cutaneous wound-healing phenotypes mediated by enhanced Areg mRNA and protein levels, we sought to identify the principal ectodomain sheddase of AREG. To this end, we generated Rhbdf2cub mice lacking ADAM17 specifically in the skin and examined the above phenotypes of Rhbdf2cub mice. We find that ADAM17 deficiency in the skin of Rhbdf2cub mice restores a full hair coat, prevents sebaceous gland enlargement, and impairs the rapid wound-healing phenotype observed in Rhbdf2cub mice. Furthermore, in vitro, stimulated shedding of AREG is abolished in Rhbdf2cub mouse embryonic keratinocytes lacking ADAM17. Thus, our data support previous findings demonstrating that ADAM17 is the major ectodomain sheddase of AREG. FEBS Open Bio 2018; 8(4):702-710

Spontaneous Posterior Segment Vascular Disease Phenotype of a Mouse Model, rnv3, Is Dependent on the Crb1rd8 Allele.

Purpose: To determine the molecular basis of lesion development in a murine model of spontaneous retinal vascularization, rnv3 (retinal vascularization 3, aka JR5558). Methods: Disease progression of rnv3 was examined in longitudinal studies by clinical evaluation, electroretinography (ERG) and light microscopy analyses. The chromosomal position for the recessive rnv3 mutation was determined by DNA pooling and genome-wide linkage analysis. The causative mutation was discovered by comparison of whole exome sequences of rnv3 mutant and wild-type (WT) controls. In order to confirm the causative mutation, transcription activator-like effector nuclease (TALEN)-mediated oligonucleotide directed repair (ODR) was utilized to correct the mutant allele. Phenotypic correction was assessed by fundus imaging and optical coherence tomography of live mice. Results: rnv3 exhibits early-onset, multifocal depigmented retinal lesions observable by fundus examination starting at 18 days of age. The retinal lesions are associated with fluorescein leakage around 25 days of age, with peak leakage at about 4 weeks of age. ERG responses deteriorate as rnv3 mutants age, concomitant with progressive photoreceptor disruption and loss that is observable by histology. Genetic analysis localized rnv3 to mouse chromosome (Chr) 1. By high throughput sequencing of a whole exome capture library of a rnv3/rnv3 mutant and subsequent sequence analysis, a single base deletion (del) in the Crb1 [crumbs family member 1] gene, which was previously reported to cause retinal degeneration 8, was identified. The TALEN-mediated ODR rescued the posterior segment vascularization phenotype; heterozygous Crb1rd8+em1Boc/Crb1rd8 and homozygous Crb1rd8+em1Boc/Crb1rd8+em1Boc mice showed a normal retinal phenotype. Additionally, six novel disruptions of Crb1 that were generated through aberrant non-homologous end joining induced by TALEN exhibited variable levels of vascularization, suggesting allelic effects. Conclusions: The rnv3 model and the models of six novel disruptions of Crb1 are all reliable, novel mouse models for the study of both early and late events associated with posterior segment vascularization and can also be used to test the effects of pharmacological targets for treating human ocular vascular disorders. Further study of these models may provide a greater understanding about how different Crb1 alleles result in aberrant angiogenesis

Inactive rhomboid proteins RHBDF1 and RHBDF2 (iRhoms): a decade of research in murine models.

Rhomboid proteases, first discovered in Drosophila, are intramembrane serine proteases. Members of the rhomboid protein family that are catalytically deficient are known as inactive rhomboids (iRhoms). iRhoms have been implicated in wound healing, cancer, and neurological disorders such as Alzheimer\u27s and Parkinson\u27s diseases, inflammation, and skin diseases. The past decade of mouse research has shed new light on two key protein domains of iRhoms-the cytosolic N-terminal domain and the transmembrane dormant peptidase domain-suggesting new ways to target multiple intracellular signaling pathways. This review focuses on recent advances in uncovering the unique functions of iRhom protein domains in normal growth and development, growth factor signaling, and inflammation, with a perspective on future therapeutic opportunities

Role of MicroRNA in Inflammatory Bowel Disease: Clinical Evidence and the Development of Preclinical Animal Models.

The dysregulation of microRNA (miRNA) is implicated in cancer, inflammation, cardiovascular disorders, drug resistance, and aging. While most researchers study miRNA\u27s role as a biomarker, for example, to distinguish between various sub-forms or stages of a given disease of interest, research is also ongoing to utilize these small nucleic acids as therapeutics. An example of a common pleiotropic disease that could benefit from miRNA-based therapeutics is inflammatory bowel disease (IBD), which is characterized by chronic inflammation of the small and large intestines. Due to complex interactions between multiple factors in the etiology of IBD, development of therapies that effectively maintain remission for this disease is a significant challenge. In this review, we discuss the role of dysregulated miRNA expression in the context of clinical ulcerative colitis (UC) and Crohn\u27s disease (CD)-the two main forms of IBD-and the various preclinical mouse models of IBD utilized to validate the therapeutic potential of targeting these miRNA. Additionally, we highlight advances in the development of genetically engineered animal models that recapitulate clinical miRNA expression and provide powerful preclinical models to assess the diagnostic and therapeutic promise of miRNA in IBD

RHBDF2-regulated growth factor signaling in a rare human disease tylosis with esophageal cancer: What can we learn from murine models?

Tylosis with esophageal cancer syndrome (TOC) is a rare autosomal dominant proliferative skin disease caused by missense mutations in the rhomboid 5 homolog 2 (RHBDF2) gene. TOC is characterized by thickening of the skin in the palms and feet and is strongly linked with the development of esophageal squamous cell carcinoma. Murine models of human diseases have been valuable tools for investigating the underlying genetic and molecular mechanisms of a broad range of diseases. Although current mouse models do not fully recapitulate all aspects of human TOC, and the molecular mechanisms underlying TOC are still emerging, the available mouse models exhibit several key aspects of the disease, including a proliferative skin phenotype, a rapid wound healing phenotype, susceptibility to epithelial cancer, and aberrant epidermal growth factor receptor (EGFR) signaling. Furthermore, we and other investigators have used these models to generate new insights into the causes and progression of TOC, including findings suggesting a tissue-specific role of the RHBDF2-EGFR pathway, rather than a role of the immune system, in mediating TOC; and indicating that amphiregulin, an EGFR ligand, is a functional driver of the disease. This review highlights the mouse models of TOC available to researchers for use in investigating the disease mechanisms and possible therapies, and the significance of genetic modifiers of the disease identified in these models in delineating the underlying molecular mechanisms

Genes adapt to outsmart gene-targeting strategies in mutant mouse strains by skipping exons to reinitiate transcription and translation.

BACKGROUND: Gene disruption in mouse embryonic stem cells or zygotes is a conventional genetics approach to identify gene function in vivo. However, because different gene disruption strategies use different mechanisms to disrupt genes, the strategies can result in diverse phenotypes in the resulting mouse model. To determine whether different gene disruption strategies affect the phenotype of resulting mutant mice, we characterized Rhbdf1 mouse mutant strains generated by three commonly used strategies-definitive-null, targeted knockout (KO)-first, and CRISPR/Cas9. RESULTS: We find that Rhbdf1 responds differently to distinct KO strategies, for example, by skipping exons and reinitiating translation to potentially yield gain-of-function alleles rather than the expected null or severe hypomorphic alleles. Our analysis also revealed that at least 4% of mice generated using the KO-first strategy show conflicting phenotypes. CONCLUSIONS: Exon skipping is a widespread phenomenon occurring across the genome. These findings have significant implications for the application of genome editing in both basic research and clinical practice

Detection of CRISPR-mediated genome modifications through altered methylation patterns of CpG islands.

BACKGROUND: The development and application of CRISPR technologies for the modification of the genome are rapidly expanding. Advances in the field describe new CRISPR components that are strategically engineered to improve the precision and reliability of CRISPR editing within the genome sequence. Genome modification using induced genome breaks that are targeted and mediated by CRISPR components leverage cellular mechanisms for repair like homology directed repair (HDR) to incorporate genomic edits with increased precision. RESULTS: In this report, we describe the gain of methylation at typically hypomethylated CpG island (CGI) locations affected by the CRISPR-mediated incorporation of donor DNA using HDR mechanisms. With characterization of CpG methylation patterns using whole genome bisulfite sequencing, these CGI methylation disruptions trace the insertion of the donor DNA during the genomic edit. These insertions mediated by homology-directed recombination disrupt the generational methylation pattern stability of the edited CGI within the cells and their cellular lineage within the animal strain, persisting across generations. Our approach describes a statistically based workflow for indicating locations of modified CGIs and provides a mechanism for evaluating the directed modification of the methylome of the affected CGI at the CpG-level. CONCLUSIONS: With advances in genome modification technology comes the need to detect the level and persistence of methylation change that modifications to the genomic sequence impose upon the collaterally edited methylome. Any modification of the methylome of somatic or germline cells could have implications for gene regulation mechanisms governed by the methylation patterns of CGI regions in the application of therapeutic edits of more sensitively regulated genomic regions. The method described here locates the directed modification of the mouse epigenome that persists over generations. While this observance would require supporting molecular observations such as direct sequence changes or gene expression changes, the observation of epigenetic modification provides an indicator that intentionally directed genomic edits can lead to collateral, unintentional epigenomic changes post modification with generational persistence