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

摘要 对人类基因组进行排序、解释和更改的能力已经改变了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 很少治愈。然而，迅速发展的基因组工程领域已经承诺了永久的、治愈的 多种遗传性疾病的选择，如代谢性肝病、大疱性表皮松解症和镰刀状 细胞疾病。操纵基因组的能力也带来了更好的疾病模型、更强大的控制 关于细胞命运，以及胚胎发育过程中细胞动力学的高分辨率地图。事实上，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并不完善，仍在开发中。还有增加编辑的空间 效率，减少偏离目标的影响，并提高目标保真度。 Cas9和腺相关病毒-6(AAV6)的结合已被证明是高效的部位特异性 基因组编辑。Cas9在靶基因组位置诱导双链断裂，而AAV6则提供单链断裂 将DNA修复模板搁置到细胞核中。由于AAV是一种病毒，它已经进化成将DNA运送到细胞中 以一种比大多数其他转染法更有效的方式进行核移植。然后，细胞利用其内源性 同源定向修复机制修复Cas9诱导的断裂，使用AAV6传递的DNA作为修复 模板。这种方法已经被用来在细胞基因组中进行小的改变和大的插入 在体外和体内。出于这些原因，AAV是全球100多项临床试验的首选媒介。 我们最近生成的数据质疑在使用AAV6进行目标位置基因组修改时的保真度 交付维修模板。使用一种全面而灵敏的方法来检测DNA的区域，我们发现 近一半的编辑细胞具有额外的、意想不到的模板基因组插入。进一步分析 据透露，这些插入是对准目标的，本质上是串联的。令人震惊的是，这种情况的频率 非预期的基因型别在文献中未见报道。研究人员和临床医生的常用技术 用于分析AAV6诱导的敲门信号将无法检测到这些串联插入。然而，还有 一些出版物中的证据，作者不知道的，支持我们的发现。 在靶向基因组编辑期间以如此高的频率发生的意外串联插入可能 会带来灾难性的后果。如果基因组修改在不知不觉中是错误的，研究人员将报告 不可靠和不正确的结果，而临床医生可能正在扰乱他们试图修复的基因。 因此，在这项建议中，我们的目标是(1)确定Cas9/AAV6诱导的连锁反应的变异和程度 与细胞类型和基因组位置有关的插入，以及(2)制定预防、减弱和 利用这些意外的串联插入。

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.