Luqman Muhammad W, Jenjaroenpun Piroon, Spathos Jessica, Shingte Nikhil, Cummins Mitchell, Nimsamer Pattaraporn, Ittner Lars M, Wongsurawat Thidathip, Delerue Fabien
Dementia Research Centre, Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, Sydney, NSW, Australia.
Khyber Medical University, Institute of Medical Sciences, Kohat, Pakistan.
Front Genome Ed. 2025 Jun 4;7:1582097. doi: 10.3389/fgeed.2025.1582097. eCollection 2025.
Over the last decade CRISPR gene editing has been successfully used to streamline the generation of animal models for biomedical research purposes. However, one limitation to its use is the potential occurrence of on-target mutations that may be detrimental or otherwise unintended. These bystander mutations are often undetected using conventional genotyping methods. The use of Adeno-Associated Viruses (AAVs) to bring donor templates in zygotes is currently being deployed by transgenic cores around the world to generate knock-ins with large transgenes (i.e., 1-4 kb payloads). Thanks to a high level of efficiency and the relative ease to establish this technique, it recently became a method of choice for transgenic laboratories. However, a thorough analysis of the editing outcomes following this method is yet to be developed. To this end, we generated three different types of integration using AAVs in two different murine genes (i.e., and ) and employed Oxford Nanopore Technologies long read sequencing to analyze the outcomes. Using a workflow that includes Cas9 enrichment and adaptive sampling, we showed that unintended on-target mutations, including duplication events and integration of viral sequences (sometimes reported using other workflows) can occur when using AAVs. This work highlights the importance of in-depth validation of the mutant lines generated and informs the uptake of this new method.
在过去十年中，CRISPR基因编辑已成功用于简化生物医学研究目的动物模型的构建。然而，其应用的一个局限性是可能出现对靶点有害或意外的突变。使用传统基因分型方法往往无法检测到这些旁观者突变。目前，世界各地的转基因核心实验室正在采用腺相关病毒（AAV）将供体模板导入受精卵，以产生携带大转基因（即1 - 4 kb有效载荷）的基因敲入。由于该技术具有较高的效率且相对容易建立，它最近成为转基因实验室的首选方法。然而，尚未对该方法后的编辑结果进行全面分析。为此，我们在两个不同的小鼠基因（即 和 ）中使用AAV产生了三种不同类型的整合，并采用牛津纳米孔技术长读长测序来分析结果。通过包括Cas9富集和适应性采样的工作流程，我们表明使用AAV时可能会出现意外的对靶点突变，包括重复事件和病毒序列整合（有时使用其他工作流程报告）。这项工作突出了对所产生突变系进行深入验证的重要性，并为采用这种新方法提供了参考。
