The Gene Sculpt Suite: a set of tools for genome editing

The discovery and development of DNA-editing nucleases (Zinc Finger Nucleases, TALENs, CRISPR/Cas systems) has given scientists the ability to precisely engineer or edit genomes as never before. Several different platforms, protocols and vectors for precision genome editing are now available, leading to the development of supporting web-based software. Here we present the Gene Sculpt Suite (GSS), which comprises three tools: (i) GTagHD, which automatically designs and generates oligonucleotides for use with the GeneWeld knock-in protocol; (ii) MEDJED, a machine learning method, which predicts the extent to which a double-stranded DNA break site will utilize the microhomology-mediated repair pathway; and (iii) MENTHU, a tool for identifying genomic locations likely to give rise to a single predominant microhomology-mediated end joining allele (PreMA) repair outcome. All tools in the GSS are freely available for download under the GPL v3.0 license and can be run locally on Windows, Mac and Linux systems capable of running R and/or Docker. The GSS is also freely available online at www.genesculpt.org

CAR T-Cell Immunotherapy in Human and Veterinary Oncology: Changing the Odds Against Hematological Malignancies

International audienceThe advent of the genome editing era brings forth the promise of adoptive cell transfer usingengineered chimeric antigen receptor (CAR) T-cells for targeted cancer therapy. CAR T-cellimmunotherapy is probably one of the most encouraging developments for the treatment ofhematological malignancies. In 2017, two CAR T-cell therapies were approved by the U. S Food andDrug Administration; one for the treatment of pediatric Acute Lymphoblastic Leukemia (ALL), the otherfor adult patients with advanced lymphomas. However, despite significant progress in the area, CART-cell therapy is still in its early days and faces significant challenges, including the complexity andcosts associated with the technology. B-cell lymphoma is the most common hematopoietic cancer indogs, with an incidence approaching 0.1% and a total of 20-100 cases per 100,000 individuals. It is awidely accepted naturally occurring model for human non-Hodgkin’s lymphoma. Current treatment iswith combination chemotherapy protocols, which prolong life for less than a year in canines and areassociated with severe dose-limiting side effects, such as gastrointestinal and bone marrow toxicity.To date, one canine study generated CAR T-cells by transfection of mRNA for CAR domainexpression. While this was shown to provide a transient anti-tumor activity, results were modest,indicating that stable, genomic integration of CAR modules is required in order to achieve lastingtherapeutic benefit. This Commentary summarizes the current state of knowledge on CAR T-cellimmunotherapy in human medicine and its potential applications in animal health, while discussingthe potential of the canine model as a translational system for immuno-oncology research

GeneWeld: a method for efficient targeted integration directed by short homology

Choices for genome engineering and integration involve high efficiency with little or no target specificity or high specificity with low activity. Here, we describe a targeted integration strategy, called GeneWeld, and a vector series for gene tagging, pGTag (plasmids for Gene Tagging), which promote highly efficient and precise targeted integration in zebrafish embryos, pig fibroblasts, and human cells utilizing the CRISPR/Cas9 system. Our work demonstrates that in vivo targeting of a genomic locus of interest with CRISPR/Cas9 and a donor vector containing as little as 24 to 48 base pairs of homology directs precise and efficient knock-in when the homology arms are exposed with a double strand break in vivo. Given our results targeting multiple loci in different species, we expect the accompanying protocols, vectors, and web interface for homology arm design to help streamline gene targeting and applications in CRISPR compatible systems

Using zebrafish to iterate and expand the precision genome writing toolbox

Designer nucleases permit the direction of DNA double-strand breaks and induction of DNA repair activities at virtually any genomic locus. The outcome of DNA repair is often non-random based on the structure of the double-strand DNA break and any homologies flanking the broken DNA ends. When an exogenous donor DNA molecule containing homology to a genomic DNA double-strand break site is supplied, DNA repair mechanisms can be hijacked to integrate the donor into the genome. However, to date rates of donor DNA integration are relatively low and recovery of precise gene targeting events is inefficient in vivo. Here, I demonstrate in zebrafish that liberating a donor cassette with CRISPR/Cas9 and the use of homologies as short as 12, 24, or 48 base pairs flanking double-stranded DNA donors can bias repair towards a Homology Directed Repair sub-pathway likely using strand annealing, herein called Homology-Mediated End Joining. This method, dubbed GeneWeld, drives precise integration in 50% of injected animals on average across 11 targeted loci and these events show low mosaicism in somatic tissue. GeneWeld events were recovered through the germline at an average rate of 50% across these loci. Southern blots demonstrate recovery of single copy, precise integration of donor cassettes at rates reasonable for average zebrafish labs, though some events are not precise. GeneWeld outperforms general Homologous Recombination (HR) in pig and human cells for integrating double-stranded DNA reporters. In addition, I apply an alternate, novel nuclease system from the CRISPR/Cas12a family, called CRISPR/Mad7, to GeneWeld in zebrafish and human cells, expanding genomic access and the genome editing toolbox. In the final part of this thesis, I describe genomic reporters that will be used to examine the genetic mechanisms of DNA repair using strand annealing in zebrafish. The work herein describes how short homologies can direct exogenous DNA integration effectively using the new method GeneWeld. GeneWeld increases accessibility and recovery of engineered genomes for functional genomics, agricultural engineering, therapeutic intervention, and addresses critical needs in the field of precision genome engineering.</p

Retinoblastoma binding protein 4 maintains cycling neural stem cells and prevents DNA damage and Tp53-dependent apoptosis in rb1 mutant neural progenitors

Retinoblastoma-binding protein 4 (Rbbp4) is a WDR adaptor protein for multiple chromatin remodelers implicated in human oncogenesis. Here we show Rbbp4 is overexpressed in zebrafish rb1-embryonal brain tumors and is upregulated across the spectrum of human embryonal and glial brain cancers. We demonstrate in vivo Rbbp4 is essential for zebrafish neurogenesis and has distinct roles in neural stem and progenitor cells. rbbp4 mutant neural stem cells show delayed cell cycle progression and become hypertrophic. In contrast, rbbp4 mutant neural precursors accumulate extensive DNA damage and undergo programmed cell death that is dependent on Tp53 signaling. Loss of Rbbp4 and disruption of genome integrity correlates with failure of neural precursors to initiate quiescence and transition to differentiation. rbbp4; rb1 double mutants show that survival of neural precursors after disruption of Rb1 is dependent on Rbbp4. Elevated Rbbp4 in Rb1-deficient brain tumors might drive proliferation and circumvent DNA damage and Tp53-dependent apoptosis, lending support to current interest in Rbbp4 as a potential druggable target.This is a pre-print made available through bioRxiv, doi: 10.1101/427344.</p

Gene Editing and Gene Therapy: Entering Uncharted Territory in Veterinary Oncology

With rapid advances in gene editing and gene therapy technologies, the development of genetic, cell, or protein-based cures to disease are no longer the realm of science fiction but that of today’s practice. The impact of these technologies are rapidly bringing them to the veterinary market as both enhanced therapeutics and towards modeling their outcomes for translational application. Simply put, gene editing enables scientists to modify an organism’s DNA a priori through the use of site-specific DNA targeting tools like clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9). Gene therapy is a broader definition that encompasses the addition of exogenous genetic materials into specific cells to correct a genetic defect. More precisely, the U.S Food and Drug Administration (FDA) defines gene therapy as “a technique that modifies a person’s genes to treat or cure disease” by either (i) replacing a disease-causing gene with a healthy copy of the gene; (ii) inactivating a disease-causing gene that was not functioning properly; or (iii) introducing a new or modified gene into the body to help treat a disease. In some instances, this can be accomplished through direct transfer of DNA or RNA into target cells of interest or more broadly through gene editing. While gene therapy is possible through the simple addition of genetic information into cells of interest, gene editing allows the genome to be reprogrammed intentionally through the deletion of diseased alleles, reconstitution of wild type sequence, or targeted integration of exogenous DNA to impart new function. Cells can be removed from the body, altered, and reinfused, or edited in vivo. Indeed, manufacturing and production efficiencies in gene editing and gene therapy in the 21st century has brought the therapeutic potential of in vitro and in vivo reprogrammed cells, to the front lines of therapeutic intervention (Brooks et al., 2016). For example, CAR-T cell therapy is revolutionizing hematologic cancer care in humans and is being translated to canines by us and others, and gene therapy trials are ongoing for mitral valve disease in dogs.This is a preprint of the article Wierson, Wesley, Alex Abel, Elizabeth Siegler, Stephen Ekker, Chad Johannes, Saad Kenderian, and Jonathan Mochel. "Gene Editing and Gene Therapy: Entering Uncharted Territory in Veterinary Oncology." Preprints (2021): 2021050376. DOI: 10.20944/preprints202105.0376.v1. Posted with permission.</p

Epigenetic regulators Rbbp4 and Hdac1 are overexpressed in a zebrafish model of RB1 embryonal brain tumor, and are required for neural progenitor survival and proliferation

In this study, we used comparative genomics and developmental genetics to identify epigenetic regulators driving oncogenesis in a zebrafish retinoblastoma 1 (rb1) somatic-targeting model of RB1 mutant embryonal brain tumors. Zebrafish rb1 brain tumors caused by TALEN or CRISPR targeting are histologically similar to human central nervous system primitive neuroectodermal tumors (CNS-PNETs). Like the human oligoneural OLIG2+/SOX10+ CNS-PNET subtype, zebrafish rb1 tumors show elevated expression of neural progenitor transcription factors olig2, sox10, sox8b and the receptor tyrosine kinase erbb3a oncogene. Comparison of rb1 tumor and rb1/rb1 germline mutant larval transcriptomes shows that the altered oligoneural precursor signature is specific to tumor tissue. More than 170 chromatin regulators were differentially expressed in rb1 tumors, including overexpression of chromatin remodeler components histone deacetylase 1 (hdac1) and retinoblastoma binding protein 4(rbbp4). Germline mutant analysis confirms that zebrafish rb1, rbbp4 and hdac1 are required during brain development. rb1 is necessary for neural precursor cell cycle exit and terminal differentiation, rbbp4 is required for survival of postmitotic precursors, and hdac1 maintains proliferation of the neural stem cell/progenitor pool. We present an in vivo assay using somatic CRISPR targeting plus live imaging of histone-H2A.F/Z-GFP fusion protein in developing larval brain to rapidly test the role of chromatin remodelers in neural stem and progenitor cells. Our somatic assay recapitulates germline mutant phenotypes and reveals a dynamic view of their roles in neural cell populations. Our study provides new insight into the epigenetic processes that might drive pathogenesis in RB1 brain tumors, and identifies Rbbp4 and its associated chromatin remodeling complexes as potential target pathways to induce apoptosis in RB1 mutant brain cancer cells.This article is published as Schultz, Laura E., Jeffrey A. Haltom, Maira P. Almeida, Wesley A. Wierson, Staci L. Solin, Trevor J. Weiss, Jordan A. Helmer, Elizabeth J. Sandquist, Heather R. Shive, and Maura McGrail. "Epigenetic regulators Rbbp4 and Hdac1 are overexpressed in a zebrafish model of RB1 embryonal brain tumor, and are required for neural progenitor survival and proliferation." Disease models & mechanisms 11, no. 6 (2018): dmm034124. doi: 10.1242/dmm.034124.</p