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

摘要 测序、解释和改变人类基因组的能力改变了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永久，治疗， 多种遗传疾病的选择，如代谢性肝病、大疱性表皮病和镰状 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 以及胚胎发育过程中细胞动力学的高分辨率地图。事实上， 所有生物科学和生物医学领域都从基因组工程的进展中受益匪浅。然而，在这方面， 用于修改基因组的工具并不完善，仍在开发中。有增加编辑的空间 效率，减少脱靶效应，并提高对靶保真度。 Cas9和腺相关病毒-6（AAV 6）的组合已被证明对于位点特异性免疫应答是高效的。 基因组编辑Cas9在靶基因组位点诱导双链断裂，而AAV 6递送单链断裂。 DNA修复模板进入细胞核。由于AAV是一种病毒，它已经进化到将DNA递送到细胞中， 以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，以使用AAV 6递送的DNA作为修复， template.这种方法已被用于在细胞基因组中进行小的改变和大的插入 在体外和体内。由于这些原因，AAV是全球100多项临床试验的首选载体。 我们最近生成的数据质疑当使用AAV 6来修饰靶位点基因组时的保真度。 交付修复模板。使用一种全面而灵敏的检测DNA区域的方法，我们发现 几乎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 揭示了这些插入在性质上是靶向的和多联体的。令人震惊的是， 非预期基因型尚未在文献中报道。常用技术研究人员和临床医生 用于分析AAV 6诱导的敲入将不能检测这些多联体插入。不过有 一些出版物中的证据，作者不知道，支持我们的发现。 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 会带来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，在该提议中，我们的目标是（1）鉴定Cas9/AAV 6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，和（2）开发预防，减弱， 利用这些无意的串联插入。

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.