Martínez‐Gálvez, Gabriel

Nearly 90% of human pathogenic mutations are caused by small genetic variations, and methods to correct these errors efficiently are critically important. One way to make small DNA changes is providing a single-stranded oligo deoxynucleotide (ssODN) containing an alteration coupled with a targeted double-strand break (DSB) at the target locus in the genome. Coupling an ssODN donor with a CRISPR-Cas9-mediated DSB is one of the most streamlined approaches to introduce small changes. However, in many systems, this approach is inefficient and introduces imprecise repair at the genetic junctions. We herein report a technology that uses spatiotemporal localization of an ssODN with CRISPR-Cas9 to improve gene alteration. We show that by fusing an ssODN template to the trans-activating RNA (tracrRNA), we recover precise genetic alterations, with increased integration and precision in vitro and in vivo . Finally, we show that this technology can be used to enhance gene conversion with other gene editing tools such as transcription activator like effector nucleases.

Mayo Clinic annually trains over 1000 predoctoral students and postdoctoral research fellows in medicine and biomedical sciences. However, training opportunities are limited for those interested in teaching and mentoring methodologies. Therefore, Mayo Clinic predoctoral students sought to provide trainee‐led learning opportunities for fellow trainees. A survey was sent to all Mayo Clinic predoctoral and postdoctoral trainees to determine preferences for pedagogy education formats. In response to survey results, the Twin Talk Series was developed. A Twin Talk is a set of one‐hour talks comprised of a Take‐in session and one or more Try‐out session(s). At the Take‐in session, a content expert discusses a pedagogical technique, and at the following Try‐out session(s), trainee volunteers practice the technique among themselves. Eight Twin Talks have been organized to date on several teaching techniques and pedagogical development topics. The impact of each Twin Talk, including understanding, format, and interest level, was evaluated through survey tools. Survey results suggested trainees favored in‐person pedagogy expert presenters during the Take‐in session over video conferences. Furthermore, modeling of the teaching technique by the expert presenter during the Take‐in session was well‐received. However, participation in the follow‐up Try‐out practice session was limited. Overall, the Twin Talk Series is a promising method for trainee‐led, peer‐to‐peer pedagogy education, particularly at institutions with limited access to pedagogical resources. The Twin Talk Series will continue to be refined to increase trainee participation, particularly in the follow‐up Try‐out sessions, and to provide relevant opportunities for trainees to practice teaching. Support or Funding Information Mayo Foundation of Medical Education and Research This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Cervical spinal hemisection at C2 (SH) paralyzes the diaphragm muscle by interrupting descending bulbospinal pathways to phrenic motoneurons. Signaling via the neurotrophin receptor TrkB in phrenic motoneurons enhances functional recovery after SH. However, it is not clear if the delay in gene expression resulting from adeno‐associated virus (AAV)‐mediated delivery would blunt the therapeutic efficacy of AAV delivered after SH. The goal of the present study was to examine the efficacy of intrapleurally‐delivered AAV7 encoding the full‐length TrkB receptor at 3 days post‐SH. Chronic diaphragm EMG recordings were conducted in adult male Sprague‐Dawley rats confirming absence of ipsilateral activity 3 days after SH. The proportion of animals displaying recovery of ipsilateral diaphragm EMG activity increased over time post‐SH and was greater in those treated with AAV‐TrkB (by 14 days post‐SH: AAV‐TrkB: 10/15 vs. AAV‐GFP: 2/11; p=0.01). Furthermore, peak root‐mean‐square amplitude of the diaphragm EMG activity increased progressively over time post‐SH. During eupnea, animals displaying recovery of ipsilateral diaphragm EMG activity that were treated with AAV‐TrkB achieved 40% of pre‐injury values (vs. 10% in those treated with AAV‐GFP). Targeting phrenic motoneurons via intrapleural delivery of AAV7‐TrkB promotes recovery of diaphragm activity when administered 3 days post‐injury. Grant Funding Source : Supported by R01 HL96750, T32 HL105355 and Mayo Clinic Center for Regenerative Medicine

Abstract Gene-editing experiments commonly elicit the error-prone non-homologous end joining for DNA double-strand break (DSB) repair. Microhomology-mediated end joining (MMEJ) can generate more predictable outcomes for functional genomic and somatic therapeutic applications. We compared three DSB repair prediction algorithms – MENTHU, inDelphi, and Lindel – in identifying MMEJ-repaired, homogeneous genotypes (PreMAs) in an independent dataset of 5,885 distinct Cas9-mediated mouse embryonic stem cell DSB repair events. MENTHU correctly identified 46% of all PreMAs available, a ∼2- and ∼60-fold sensitivity increase compared to inDelphi and Lindel, respectively. In contrast, only Lindel correctly predicted predominant single-base insertions. We report the new algorithm MENdel, a combination of MENTHU and Lindel, that achieves the most predictive coverage of homogeneous out-of-frame mutations in this large dataset. We then estimated the frequency of Cas9-targetable homogeneous frameshift-inducing DSBs in vertebrate coding regions for gene discovery using MENdel. 47 out of 54 genes (87%) contained at least one early frameshift-inducing DSB and 49 out of 54 (91%) did so when also considering Cas12a-mediated deletions. We suggest that the use of MENdel helps researchers use MMEJ at scale for reverse genetics screenings and with sufficient intra-gene density rates to be viable for nearly all loss-of-function based gene editing therapeutic applications.

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.

Genome engineering has gone mainstream because of breakthroughs in defining and harnessing naturally occurring, customizable DNA recognition cursors (protein or RNA-guided). At present, most gene editing relies on these cursors to direct custom DNA endonucleases to a specific genomic sequence to induce a double-strand break. New tools for genome engineering are continuously being explored, and another advance in DNA targeting has recently been described. Argonaute isolated from Natronobacterium gregoryi (NgAgo) is an ssDNA-based cursor that thus far has no known limitations in sequence recognition, shows promise for high specificity, and for many applications may represent a potentially more accessible genome-editing system over prior tools as it requires only a single, 24-base, 5′ phosphorylated ssDNA for DNA targeting. Genome engineering is in a remarkable moment of unprecedented growth with exponential reduction in costs reminiscent of Moore's law in electronics. Many questions remain with regard to Argonaute utility in specific systems, but there is no doubt that genome engineering is expanding into new and exciting areas from synthetic biology to gene therapy.

Transcription activator-like effectors (TALEs) are extremely effective, single-molecule DNA-targeting molecular cursors used for locus-specific genome science applications, including high-precision molecular medicine and other genome engineering applications. TALEs are used in genome engineering for locus-specific DNA editing and imaging, as artificial transcriptional activators and repressors, and for targeted epigenetic modification. TALEs as nucleases (TALENs) are effective editing tools and offer high binding specificity and fewer sequence constraints toward the targeted genome than other custom nuclease systems. One bottleneck of broader TALE use is reagent accessibility. For example, one commonly deployed method uses a multitube, 5-day assembly protocol. Here we describe FusX, a streamlined Golden Gate TALE assembly system that (1) is backward compatible with popular TALE backbones, (2) is functionalized as a single-tube 3-day TALE assembly process, (3) requires only commonly used basic molecular biology reagents, and (4) is cost-effective. More than 100 TALEN pairs have been successfully assembled using FusX, and 27 pairs were quantitatively tested in zebrafish, with each showing high somatic and germline activity. Furthermore, this assembly system is flexible and is compatible with standard molecular biology laboratory tools, but can be scaled with automated laboratory support. To demonstrate, we use a highly accessible and commercially available liquid-handling robot to rapidly and accurately assemble TALEs using the FusX TALE toolkit. Together, the FusX system accelerates TALE-based genomic science applications from basic science screening work for functional genomics testing and molecular medicine applications.