Ekker, Stephen C.

Purpose Hearing loss is a common impairment of the human senses with an estimated 48 million American adults reporting some trouble hearing; however, access to hearing health care is limited. Detection of hearing loss through a mobile, handheld tool can provide an important access point and potentially expedited access to the continuum of hearing health care. Here, we determined that GoAudio, a portable, automated hearing assessment tool, can be used to identify individuals who require additional hearing evaluation in a clinical workflow. Method This initial study included 24 adults, ages 18–65 years ( M = 50, SD = 12), tested with GoAudio versus “gold-standard” clinical audiometry for eight frequencies to evaluate “real-world” applications. Participants utilized noise-canceling headphones combined with a tablet-based application for the GoAudio assessment. Results The primary study outcome measurements were the comparison of hearing thresholds (dB HL) from clinical audiometry and GoAudio. Results suggest that GoAudio is comparable to clinical audiometry for the identification of hearing loss at most frequencies (except 1 kHz for both ears and 2 kHz in the right ear). Upon stratifying data based on age, we identified that GoAudio is capable of identifying suspected age-related hearing loss or hearing thresholds greater than 30 dB HL at higher frequencies in both ears. Conclusion The study results support that GoAudio can be used effectively in clinical practice workflows as a reliable hearing assessment tool for the identification of hearing loss at the majority of frequencies outside a sound-treated booth and can detect characteristics of age-related hearing loss. Supplemental Material https://doi.org/10.23641/asha.13087682

One key bottleneck in understanding the human genome is the relative under-characterization of 90% of protein coding regions. We report a collection of 1200 transgenic zebrafish strains made with the gene-break transposon (GBT) protein trap to simultaneously report and reversibly knockdown the tagged genes. Protein trap-associated mRFP expression shows previously undocumented expression of 35% and 90% of cloned genes at 2 and 4 days post-fertilization, respectively. Further, investigated alleles regularly show 99% gene-specific mRNA knockdown. Homozygous GBT animals in ryr1b , fras1 , tnnt2a , edar and hmcn1 phenocopied established mutants. 204 cloned lines trapped diverse proteins, including 64 orthologs of human disease-associated genes with 40 as potential new disease models. Severely reduced skeletal muscle Ca 2+ transients in GBT ryr1b homozygous animals validated the ability to explore molecular mechanisms of genetic diseases. This GBT system facilitates novel functional genome annotation towards understanding cellular and molecular underpinnings of vertebrate biology and human disease.

Abstract Colon polyps are the most common neoplastic finding at colonoscopy and have the potential to transform to cancer. Understanding the mechanisms involved in the transformation from normal colon to polyp is central to the identification of interventions to prevent or stop a polyp from forming or, in the worst case, from transforming. Therefore, the clinical conundrum is to predict which polyps will progress. It is crucial to understand if there are molecular features that define premalignant lesions and that may contribute to the final steps of transformation, which could potentially be targets for early detection or more individualized clinical management. Cellular transformation is driven by the interplay of genetic and epigenetic changes, and a large body of research has identified modifications of chromatin and the role of those modifications in regulating genes. Telomeres, telomere maintenance mechanisms, and chromatin remodelers maintain genetic stability, and their disruption is associated with the development of many diseases, including cancer. Organoids are multicellular structures grown from the stem cells of an organ to recapitulate the diverse cell types of the parent organ. This renewable resource of an individual’s tissue provides the opportunity to study colon carcinogenesis in combination with advanced genomic tools such as gene editing. The overall objective of this study is to determine the role of telomere and chromatin remodeler alterations in neoplastic transformation. The functional interaction between hTERT and specific chromatin remodelers will be resolved in two steps: 1) activating telomerase in normal colon and polyp organoids from patients and measuring chromatin remodeler expression, and 2) incorporating mutations in the chromatin remodelers using genome editing and then measuring the expression of hTERT in the normal colon and polyp organoids from patients with cancer-adjacent and cancer-free polyps. Gene editing experiments were performed utilizing transposon-based vectors (PiggyBac), short homology arm-based knockin (GeneWeld), or CRISPR/Cas9, coupled with electroporation for cargo delivery. To assess neoplastic transformation as a result of the gene edits, we used histology and cellular morphology of the organoid specimen by hematoxylin and eosin staining and pathologist review, and the markers by immunofluorescence. These models of neoplastic transformation in organoids from patient tissues provide an accessible tool to visualize the functional changes that correspond to alterations in telomerase and chromatin remodelers and how alterations in these targets affect growth, proliferation, differentiation, and cell turnover. Identification of epigenomic molecular alterations linked with progression of polyps to cancer could lead to modifiable targets for preventive interventions and provide candidate molecular markers in order to better tailor polyp patient screening recommendations. Note: This abstract was not presented at the conference. Citation Format: Brooke R. Druliner, Brandon Simone, Miguel Munoz-Gomez, Kim Kossick, Daniel O'Brien, Alexej Abyzov, Stephen Ekker, Lisa A. Boardman. Characterizing telomere and chromatin dynamics in colorectal cell transformation [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A33.

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24–48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22–100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.

RXRA is an endothelial- and early stage–predominant therapeutic target for treating anthracycline-induced cardiotoxicity.

CRISPR and CRISPR-Cas effector proteins enable the targeting of DNA double-strand breaks to defined loci based on a variable length RNA guide specific to each effector. The guide RNAs are generally similar in size and form, consisting of a ∼20 nucleotide sequence complementary to the DNA target and an RNA secondary structure recognized by the effector. However, the effector proteins vary in protospacer adjacent motif requirements, nuclease activities, and DNA binding kinetics. Recently, ErCas12a, a new member of the Cas12a family, was identified in Eubacterium rectale . Here, we report the first characterization of ErCas12a activity in zebrafish and expand on previously reported activity in human cells. Using a fluorescent reporter system, we show that CRISPR-ErCas12a elicits strand annealing mediated DNA repair more efficiently than CRISPR-Cas9. Further, using our previously reported gene targeting method that utilizes short homology, GeneWeld, we demonstrate the use of CRISPR-ErCas12a to integrate reporter alleles into the genomes of both zebrafish and human cells. Together, this work provides methods for deploying an additional CRISPR-Cas system, thus increasing the flexibility researchers have in applying genome engineering technologies.

By enabling researchers to image whole zebrafish with cellular resolution, X-ray histotomography will improve our understanding of the biological differences between individuals of the same species.

Abstract 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.