Jia Mary S, Padmaswari Made Harumi, Burcham Landon A, Agrawal Shilpi, Bulliard Gabrielle N, Stokes Abbey L, Nelson Christopher E
University of Arkansas, Biomedical Engineering.
Cellular and Molecular Biology.
bioRxiv. 2025 Aug 1:2025.08.01.668007. doi: 10.1101/2025.08.01.668007.
Adeno associated virus (AAV)-mediated delivery of CRISPR associated nucleases (AAV-CRISPR) is a promising solution to treat genetic diseases such as Duchenne Muscular Dystrophy (DMD) and is now in early clinical trials. However, genotoxicity and immunogenicity concerns have hindered clinical translation. Due to the complex etiology associated with DMD, the post-transduction consequences of double-stranded breaks induced by AAV-CRISPR in disease models are unclear. This barrier is partially conferred by conventional sequencing methods where common outcomes of AAV-CRISPR editing often escape detection. However, recent reports of novel long-read sequencing approaches permit comprehensive variant detection using a broader sequence context. Here, we comprehensively investigated genomic and transcriptomic post-AAV-CRISPR transduction consequences in myoblast cells and a DMD mouse model following intramuscular and intravenous AAV-CRISPR therapy using both long- and short-read sequencing techniques. Structural variant characterization indicates that unintended on-target large insertions and inversions are common editing outcomes. We demonstrate that combining adaptive sampling with nanopore Cas9-targeted sequencing (AS-nCATS) for long-read quantification of AAV integration is synergistic for detecting difficult-to-amplify editing events. This unbiased data suggests that full-length AAV integration is equally as probable as the on-target deletion. Further, we develop a Nanopore Rapid Amplification of cDNA Ends (nRACE-seq) pipeline for long-read detection of unknown 5' or 3' ends of edited transcripts. The nRACE-seq approach effectively detects the presence of AAV- chimeric transcripts, erroneous splicing events, and off-target AAV integration sites. In summary, our findings offer insights into the adaptation of AAV-CRISPR DSB-mediated therapeutics for monogenic diseases and promote the standardization of CRISPR evaluation. We highlight the importance of coupling polymerase-based and polymerase-free methods in long-read sequencing to assess editing outcomes as the field progresses toward clinical applications.
腺相关病毒（AAV）介导的CRISPR相关核酸酶递送（AAV-CRISPR）是治疗诸如杜氏肌营养不良症（DMD）等遗传疾病的一种有前景的解决方案，目前正处于早期临床试验阶段。然而，基因毒性和免疫原性问题阻碍了其临床转化。由于与DMD相关的病因复杂，疾病模型中AAV-CRISPR诱导的双链断裂的转导后后果尚不清楚。传统测序方法在一定程度上造成了这一障碍，因为AAV-CRISPR编辑的常见结果往往无法被检测到。然而，最近有关新型长读长测序方法的报道允许在更广泛的序列背景下进行全面的变异检测。在这里，我们使用长读长和短读长测序技术，全面研究了在成肌细胞和DMD小鼠模型中，肌肉内和静脉内给予AAV-CRISPR治疗后，基因组和转录组的转导后后果。结构变异特征表明，非预期的靶向大插入和倒位是常见的编辑结果。我们证明，将适应性采样与纳米孔Cas9靶向测序（AS-nCATS）相结合用于AAV整合的长读长定量，对于检测难以扩增的编辑事件具有协同作用。这些无偏差的数据表明，全长AAV整合与靶向缺失的可能性相同。此外，我们开发了一种纳米孔cDNA末端快速扩增（nRACE-seq）流程，用于长读长检测编辑转录本未知的5'或3'末端。nRACE-seq方法有效地检测到了AAV嵌合转录本的存在、错误剪接事件和脱靶AAV整合位点。总之，我们的研究结果为将AAV-CRISPR DSB介导的疗法应用于单基因疾病提供了见解，并促进了CRISPR评估的标准化。随着该领域向临床应用发展，我们强调在长读长测序中结合基于聚合酶和无聚合酶方法来评估编辑结果的重要性。
