2025 Hyper Recent •CC0 1.0 Universal

This work is dedicated to the public domain. No rights reserved.

Access Preprint From Server
January 21st, 2025
Version: 1
University of WA/Fred Hutchinson Cancer Research Center
molecular biology
biorxiv

Minimization of gene editing off-target effects by tissue restriction of expression

Kim, N.-G.Open in Google Scholar•Aubert, M.Open in Google Scholar•Haick, A. K.Open in Google Scholar•Massa, P. A.Open in Google Scholar•Loprieno, M. A.Open in Google Scholar•Walter, M.Open in Google Scholar•Kim, H. G.Open in Google Scholar•Nunley, B. E.Open in Google Scholar•Xie, H.Open in Google Scholar•Stensland, L.Open in Google Scholaret al.

Therapeutic in vivo gene editing with highly specific nucleases has the potential to revolutionize treatment for a wide range of human diseases, including genetic disorders and latent viral infections like herpes simplex virus (HSV). However, challenges regarding specificity, efficiency, delivery, and safety must be addressed before its clinical application. A key concern is the risk of off-target effects, which can cause unintended and potentially harmful genetic changes. We previously developed a curative in vivo gene editing approach to eliminate latent HSV using HSV-specific meganuclease delivered by an AAV vector. In this study, we investigate off-target effects of meganuclease by identifying potential off-target sites through GUIDE-tag analysis and assessing genetic alterations using amplicon deep sequencing in tissues from meganuclease treated mice. Our results show that meganuclease expression driven by a ubiquitous promoter leads to high off-target gene editing in the mouse liver, a non-relevant target tissue. However, restricting the meganuclease expression with a neuron-specific promoter and/or a liver-specific miRNA target sequence efficiently reduces off-target effects in both liver and trigeminal ganglia. These findings suggest that incorporation of regulatory DNA elements for tissue-specific expression in viral vectors can reduce off-target effects and improve the safety of therapeutic in vivo gene editing.

Similar Papers

biorxiv
Thu May 08 2025
WITHDRAWN: Investigation of coronaviruses, paramyxoviruses and poxviruses in free-ranging anteaters and armadillos: a search for the unknown and zoonotic implications
The authors have withdrawn this manuscript because of the necessity of correcting fundamental information. A corrected version may be submitted in the future. Therefore, the authors do not wish this work to be cited as a reference for the project. If you have any questions, please contact the corresponding author.
Martins, N. B.
•
Alves, M. H.
•
De Navasquez, L. M. L.
•
Linhares, Y. S.
...•
Hora, A. S. d.
biorxiv
Thu May 08 2025
Venomous Maggots? A first exploration of the toxin arsenal of larval stages of the horse fly Tabanus autumnalis
As a rapidly evolving trait, animal venom exhibits compositional variation even at the intraspecific level between populations, ontogenetic stages and sexes. In extreme cases venom is used for different functional purposes across ontogenetic stages. This phenomenon occurs for example in case of horse flies (Tabanidae), which utilize venom for predation during the larval stage and for blood feeding...
Kraemer, J.
•
Dersch, L.
•
Vilcinskas, A.
•
Lueddecke, T.
biorxiv
Thu May 08 2025
PU.1 and TGF-β signaling transactivate CD103 expression in mast cells and dendritic cells: Opposite roles of GATA2 in the expression of mucosal mast cell-specific genes
Mucosal mast cells (MMCs) are distinguished from connective tissue MCs by the specific expression of integrin CD103 (E/{beta}7) and MC proteases Mcpt1 and Mcpt2. Although the expression of the Mcpt1 and Mcpt2 genes is cooperatively regulated by the transcription factor GATA2 and TGF-{beta} signaling in MMCs, the transcriptional mechanism of CD103 expression remains unknown. Here, we found that sur...
Ishii, K.
•
Nagata, K.
•
Yamashita, N.
•
Yamazaki, Y.
...•
Nishiyama, C.
biorxiv
Thu May 08 2025
Molecular epidemiology of Theileria parva in Eastern Democratic Republic of Congo: Implications to the introduction of the Muguga Cocktail vaccine for East Coast Fever
East Coast Fever (ECF) is one of the most economically important tick-borne diseases of cattle in Eastern, Central, and Southern Africa, caused by the intracellular protozoan parasite Theileria parva (T. parva). This study investigated the prevalence and genetic diversity of T. parva populations in the eastern Democratic Republic of Congo (DRC) to inform immunization strategies against ECF. By emp...
Muleya, W.
•
Mulonga, S.
•
Mbao, V.
•
Kasereka, K. M.
...•
Atuhaire, D. K.
biorxiv
Wed May 07 2025
Promoter strength and position govern promoter competition
Competition between promoters within a shared regulatory landscape has been implicated in development and disease, but the determinants of promoter competition have not been systematically studied. Here, we use a synthetic platform to introduce diverse promoters at defined genomic sites within the Sox2 locus and measure how these inserted promoters attenuate activity of the endogenous promoter. We...
Koska, M.
•
Swigut, T.
•
Boettiger, A. N.
•
Wysocka, J.
biorxiv
Wed May 07 2025
Autophagy selectively clears ER in inflammation-induced muscle atrophy
Skeletal muscle atrophy is a pathological condition characterized by the progressive loss of muscle mass and function, driven by factors such as disuse, inflammation, and aging. While the ubiquitin-proteasome system is established as the central mediator of myofibrillar protein degradation, the role of autophagy in selective protein turnover remains largely unexplored. To address this, we employed...
Dueren, U. K.
•
Wei, A. A. J.
•
Gressler, A. E.
•
Popp, O.
...•
Sommer, T.
biorxiv
Wed May 07 2025
Transcriptional Roadmap of the Human Airway Epithelium Identifying HLF as a Novel Regulator of Basal Stem Cell Function
Rationale: The human airway epithelium depends on a coordinated hierarchy of stem- and differentiated cells to maintain tissue integrity and respond to injury. Defining the transcriptional and translational programs that govern these processes is critical for understanding airway disease and advancing regenerative therapies. Objectives: To map the transcriptional landscape of the human airway epit...
Prabhala, P.
•
Freiman, S.
•
Gvazava, N.
•
Sharma, J.
...•
Magnusson, M.
biorxiv
Wed May 07 2025
BLADE-R: streamlined RNA extraction for molecular diagnostics and high-throughput applications
Efficient nucleic acid extraction and purification are crucial for cellular and molecular biology research, yet they pose challenges for large-scale clinical RNA sequencing and PCR assays. Here, we present BLADE-R, a magnetic bead-based protocol that simplifies the process by combining cellular lysis and nucleic acid binding into a single step, followed by a unique on-bead rinse for nuclease-free ...
Tajammal, A.
•
Haddox, S.
•
Zahra, S.
•
Cornelison, R.
...•
Li, H.
biorxiv
Wed May 07 2025
Substitutions in RNA-binding protein Hrp1 map a potential interaction surface with the yeast RNA polymerase II elongation complex
Anti-termination factors for eukaryotic RNA polymerase II (RNAP II) that are released upon binding sequences in the terminator of nascent transcripts were proposed almost 40 years ago but few candidates have been found. Here we report genetic evidence that the yeast nuclear RNA-binding protein Hrp1, also known as Nab4 and CF1B, acts as an RNAP II anti-termination factor. A Lys to Glu substitution ...
Wang, M.
•
Arora, P.
•
Kaplan, C. D.
•
Brow, D. A.
biorxiv
Wed May 07 2025
CRISPR-Cas9 HDR Optimization: RAD52, Denatured and 5'-Modified DNA Templates in Knock-In Mice Generation
CRISPR/Cas9-mediated genome editing is a powerful tool for producing animal models of human diseases. However, it often encounters challenges related to low efficiency of donor DNA templates insertion through homology-directed repair (HDR) pathway or unwanted insertions and/or multiplications. Here, we present findings from multiple targeting experiments aimed at generating a Nup93 conditional kno...
Skryabin, B. V.
•
Braun, D. A.
•
Kaiser, H.
•
Gubar, L.
...•
Rozhdestvensky, T. S.