Knockin marmoset reporters for non-invasive measuring of genome-editing efficiency

项目摘要 在过去的十年里，基因组编辑技术取得了前所未有的进步，并批准了 美国食品和药物管理局的几种靶向基因疗法。因此，基于靶向核酸内切酶的 基因治疗的方法现在对广泛的治疗和逆转大的 人类疾病的数量。尽管CRISPR/CAS系统的早期版本存在以下问题 效率和特异性，通过定向进化，靶向蛋白质工程， 编辑增强剂的发现使基因疗法的广泛应用变得触手可及。然而， 基础科学与实施安全有效的临床干预措施之间的一个主要障碍 缺乏合适的大型动物模型来进行新技术和治疗策略的临床前测试。 在这里，我们提出了一种策略，在常见的绒猴(Callithrix Jacchus)中生成两个报告行 将用于监控Cas9和腺嘌呤基单细胞编辑人员的目标上和目标外编辑 决议。大型动物记者必须高效，能够检测基因的多个方面 编辑，如果他们要广泛使用，并能够克服缓慢的性成熟和怀孕 灵长类动物的时期。考虑到这一点，我们将利用成熟的荧光记者在 结合一种名为Akaluc的新开发的荧光素酶变体，可用于非侵入性 单细胞敏感度的绒猴生物发光成像。我们将生成一个初始集合，每组两个 能够以非侵入性方式测试敲入和腺嘌呤碱基编辑的绒猴记者 靶向编辑介导的Venus-Akaluc表达的激活。在对目标进行初步筛选后 通过使用电子倍增电荷耦合设备(EMCCD)相机进行全身成像编辑，组织可以 从这些报告动物中收集，并用于目标上和目标外编辑的单细胞分析 通过荧光激活细胞分选(FACS)和单细胞测序。我们的方法建立了一个 用于在非人类灵长类物种中测试编辑技术的通用和高效平台，并且可以 轻松、快速地适应不断发展的技术，使其成为加速 开发安全有效的基因疗法。

Project Summary The past decade has seen unprecedented advances in genome editing technologies and the approval of several targeted gene therapies by the Food & Drug Administration. As such, targeted endonuclease-based approaches to gene therapy now hold realistic promise for the widespread treatment and reversal of a large number of human diseases. Although early versions of CRISPR/Cas systems suffered from problems with efficiency and specificity, recent advances achieved through directed-evolution, targeted protein engineering, and the identification of editing enhancers have put widespread use of gene therapy within reach. However, one major hurdle between the basic science and the implementation of safe and effective clinical interventions is a lack of suitable large animal models for pre-clinical testing of new technologies and therapeutic strategies. Here, we propose a strategy to generate two reporter lines in the common marmoset (Callithrix jacchus) that will be used for monitoring of both on- and off-target editing by Cas9 and adenine base editors at single-cell resolution. Large animal reporters must be highly efficient and capable of testing multiple aspects of gene editing if they are to be broadly useful and capable of overcoming the slow sexual maturation and gestational periods of primates. Taking this into consideration, we will utilize well-established fluorescent reporters in conjunction with a newly-developed variant of luciferase called Akaluc that can be used for non-invasive bioluminescent imaging in marmosets with single-cell sensitivity. We will generate an initial set of two marmoset reporters capable of testing knock-in and adenine base editing in a non-invasive manner through on-target editing-mediated activation of Venus-Akaluc expression. Following initial screening for on-target editing via whole-body imaging with an electron-multiplying charge-couple device (EMCCD) camera, tissue can be collected from these reporter animals and used for single-cell analyses of both on- and off-target editing through fluorescence activated cell sorting (FACS) and single-cell sequencing. Our approach establishes a versatile and efficient platform for testing editing technologies in a non-human primate species and can be easily and rapidly adapted to evolving technologies, making it a valuable system for accelerating the development of safe and effective gene therapies.