Frequent concatemeric insertions during AAV6/Cas9-mediated genome editing: Detection and Prevention

AAV6/Cas9 介导的基因组编辑过程中频繁的串联插入：检测和预防

• 批准号: 10193723
• 负责人: Ravindra Majeti
• 金额: $ 23.69万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-15 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10193723
• 关键词: Animal Disease Models; Attenuated; Biological Assay; Biological Sciences; Cell Line; Cell Nucleus; Cells; Clinical Trials; Communities; DNA; DNA Repair; DNA Sequence Alteration; DNA cassette; DNA delivery; Dangerousness; Data; Dependovirus; Detection; Development; Discipline; Disease; Disease model; Embryonic Development; Engraftment; Ensure; Epidermolysis Bullosa; Excision; Frequencies; Genes; Genetic Diseases; Genome; Genome engineering; Genomic Segment; Genomics; Genotype; Hematological Disease; Hematopoietic stem cells; Human; Human Genome; In Vitro; Inverted Terminal Repeat; Knock-in; Link; Literature; Liver diseases; Location; Maps; Mediating; Medical; Metabolic; Methods; Modification; Nature; Outcome; Pluripotent Stem Cells; Prevention; Property; Protocols documentation; Publications; Publishing; Reporting; Reproducibility of Results; Research Personnel; Residual state; Resolution; Sickle Cell Anemia; Single-Stranded DNA; Site; Techniques; Testing; Transfection; Transplantation; Variant; Viral; Virus; Work; adeno-associated viral vector; cell type; clinically relevant; detection method; digital; gain of function; gene correction; genome editing; human embryonic stem cell line; human pluripotent stem cell; improved; in vivo; induced pluripotent stem cell; innovation; knockout gene; novel; particle; prevent; repaired; tool; vector

ABSTRACT The ability to sequence, interpret, and make changes to the human genome has transformed 21st century biosciences. Historically, diseases caused by genetic mutations could be at best recognized and treated, but rarely cured. However, the rapidly developing field of genome engineering has promised permanent, curative options for a multitude of genetic conditions such as metabolic liver diseases, epidermolysis bullosa, and sickle cell disease. The ability to manipulate the genome has also led to better disease models, more robust control over cellular fate, and high-resolution maps of cellular dynamics during embryonic development. Indeed, nearly all bioscientific and biomedical fields have benefited greatly from advances in genome engineering. However, the tools used to modify the genome are imperfect and still in development. There is room to increase editing efficiency, decrease off-target effects, and improve on-target fidelity. The combination of Cas9 and adeno-associated virus-6 (AAV6) has proven to be highly efficient for site-specific genome editing. Cas9 induces a double-stranded break at a target genomic site, while AAV6 delivers single- stranded DNA repair templates into the nucleus. Since AAV is a virus, it has evolved to deliver DNA into cell nuclei in a manner more efficient than most other transfection protocols. The cell then employs its endogenous homology-directed repair machinery to fix the Cas9-induced break, using the AAV6-delivered DNA as a repair template. This approach has been used to make both small changes and large insertions in the genome of cells in vitro and in vivo. For these reasons, AAV is the vector-of-choice in over 100 clinical trials worldwide. We recently generated data that questions the fidelity of target-site genome modifications when using AAV6 to deliver the repair template. Using a comprehensive and sensitive assay for detecting regions of DNA, we found that nearly half of the edited cells had additional, unexpected genomic inserts of the template. Further analysis revealed that these insertions are on-target and concatemeric in nature. Shockingly, the frequency of this unintended genotype has not been reported in the literature. Common techniques researchers and clinicians use to analyze AAV6-induced knockins would fail to detect these concatemeric insertions. However, there is evidence in some of the publications that, unknown to the authors, supports our finding. Unintended concatemeric insertions during targeted genome-editing that occur at such high frequencies could have disastrous consequences. If genomic modifications are unknowingly incorrect, researchers will report unreliable and incorrect results, while clinicians may be disrupting the genes in which they are trying to repair. Therefore, in this proposal we aim to (1) identify the variation and extent of Cas9/AAV6-induced concatemeric insertions in regards to cell-type and genomic location, and (2) develop strategies to prevent, attenuate, and exploit these unintended concatemeric insertions.

抽象的 测序、解释和改变人类基因组的能力已经改变了 21 世纪 生物科学。从历史上看，由基因突变引起的疾病最多只能被识别和治疗，但是 很少治愈。然而，快速发展的基因组工程领域有望带来永久性的、治愈性的治疗效果。 针对多种遗传疾病的选择，例如代谢性肝病、大疱性表皮松解症和镰状细胞病 细胞疾病。操纵基因组的能力也带来了更好的疾病模型和更强大的控制 细胞命运以及胚胎发育过程中细胞动力学的高分辨率图。确实，几乎 所有生物科学和生物医学领域都从基因组工程的进步中受益匪浅。然而， 用于修改基因组的工具还不完善并且仍在开发中。编辑还有增加空间 效率、减少脱靶效应并提高目标保真度。 Cas9 和腺相关病毒 6 (AAV6) 的组合已被证明对于位点特异性非常有效 基因组编辑。 Cas9 在目标基因组位点诱导双链断裂，而 AAV6 则提供单链断裂 链DNA修复模板进入细胞核。由于 AAV 是一种病毒，它已经进化到能够将 DNA 输送到细胞中 比大多数其他转染方案更有效的方式转染细胞核。然后细胞利用其内源性 同源定向修复机制，使用 AAV6 传递的 DNA 作为修复来修复 Cas9 诱导的断裂 模板。这种方法已用于在细胞基因组中进行小的改变和大的插入 体外和体内。出于这些原因，AAV 成为全球 100 多项临床试验的首选载体。 我们最近生成的数据质疑使用 AAV6 进行目标位点基因组修饰的保真度。 交付维修模板。使用全面且灵敏的检测方法来检测 DNA 区域，我们发现 近一半的编辑细胞有额外的、意想不到的模板基因组插入。进一步分析 表明这些插入本质上是定向的和串联的。令人震惊的是，这样的频率 文献中尚未报道非预期的基因型。研究人员和临床医生的常用技术 用于分析 AAV6 诱导的基因敲入将无法检测到这些串联插入。然而，有 一些出版物中作者不知道的证据支持了我们的发现。 在靶向基因组编辑过程中以如此高的频率发生的意外串联插入可能会导致 造成灾难性的后果。如果基因组修饰在不知不觉中不正确，研究人员将报告 不可靠和不正确的结果，而临床医生可能会破坏他们试图修复的基因。 因此，在本提案中，我们的目标是 (1) 确定 Cas9/AAV6 诱导的多联体的变异和程度 关于细胞类型和基因组位置的插入，以及（2）制定预防、减弱和 利用这些意外的串联插入。