Scalable Development of Custom Genome Editing Technologies

项目总结 基因组编辑技术促进了基础和应用生物医学研究的重大进展 菲尔兹。尽管CRISPR-Cas酶用于基因组编辑的适应促进了和戏剧性地 加速了编辑活细胞中核酸序列的能力，许多基因工程方法是 使用一种具有显著局限性的单一酶来完成。自然产生的CRISPR-Cas9效应器 来自细菌的化脓性链球菌(SpCas9)可以有效地工作于某些编辑应用程序，但是 对于治疗导致遗传疾病的各种序列来说，这并不是一个“一刀切”的解决方案。临床部 SpCas9的潜力由于酶的自然特性而固有地受到限制，包括对 绑定一个短的DNA基序以启动编辑。这个基序只出现在基因组的一小部分中，防止SpCas9 通过编辑许多不包含该序列的致病突变。SpCas9无法打靶 广泛的DNA站点，以及其他不太理想的特征，说明了创新的必要性 释放基因组编辑在临床上的广泛潜力。实现更全面基因组的一种解决方案 靶向是利用定向进化来设计能够识别新基序的新形式的SpCas9。 然而，传统的蛋白质工程方法仍然存在产量低、成本高和费力的问题。在这里，我们将 通过优化可扩展的方法来设计和表征，缩小这些技术和方法差距 性能得到改善的新型CaS变体。我们建议的研究将解决以下突出限制 CRISPR-CAS酶：(1)开发可扩展的实验方法，以更快、更有效地 设计和表征蛋白质，(2)优化机器学习引导的定向进化以创建目录 可定制的DNA编辑器，以及(3)作为概念验证，彻底评估PAM选择性编辑器的目录 对抗导致常见病和罕见病的突变。该项目的长期愿景是创建一个 定制的编辑目录，它们一起可以系统地定位基因组，而不会牺牲其他重要的 特性，比如专一性。为了使编辑完全大众化，这组优化的CRISPR技术将 为研究人员和临床医生创建一个虚拟的“一站式商店”，为患者寻求优化的基因治疗。 成功完成拟议的研究将使实验和计算方法相结合，将 为描述和改进基因组编辑活动提供新的可扩展的方法 技术，并将指数级扩展编辑“工具箱”中的功能。团结在一起， 开发和实施定制CA编辑器目录，并将加速和创建蓝图 将安全有效的CRISPR疗法转化为造福患者。

PROJECT SUMMARY Genome editing technologies have catalyzed major advances across basic and applied biomedical research fields. Although the adaptation of CRISPR-Cas enzymes for genome editing has facilitated and dramatically accelerated the ability to edit nucleic acid sequences in living cells, many genetic engineering approaches are performed using a single enzyme that has notable limitations. The naturally occurring CRISPR-Cas9 effector from the bacterium Streptococcus pyogenes (SpCas9) can function efficiently for certain editing applications, but is not a ‘one-size-fits-all’ solution for treating the diversity of sequences that cause genetic disorders. The clinical potential of SpCas9 is inherently limited due to the natural characteristics of the enzyme, including a requirement to bind a short DNA motif to initiate editing. This motif only occurs in a fraction of the genome, preventing SpCas9 from editing many disease-causing mutations that do not harbor this sequence. The inability of SpCas9 to target a broad range of DNA sites, along with other suboptimal characteristics, illustrates the need for innovations to unlock the wide potential of genome editing in the clinic. One solution to enable more comprehensive genome targeting is to utilize directed evolution to engineer new forms of SpCas9 that can recognize new motifs. However, traditional protein engineering approaches remain low-throughput, costly, and laborious. Here we will close these technological and methodological gaps by optimizing scalable methods to engineer and characterize novel Cas variants with improved properties. Our proposed research will address prominent limitations of CRISPR-Cas enzymes by: (1) developing scalable experimental approaches to more rapidly and effectively engineer and characterize proteins, (2) optimizing machine learning-guided directed evolution to create a catalog of customizable DNA editors, and (3) as proof-of-concept, thoroughly evaluate a catalog of PAM-selective editors against mutations that cause common and rare diseases. The long-term vision of this project is to create a catalog of bespoke editors that together can systematically target the genome without sacrificing other important properties like specificity. To fully democratize editing, this collection of optimized CRISPR technologies will create a virtual ‘one-stop-shop’ for researchers and clinicians seeking optimized genetic treatments for patients. Successful completion of the proposed studies will synergize experimental and computational methods, will provide novel scalable approaches for characterizing and improving the activities of genome editing technologies, and will exponentially expand the capabilities within the editing ‘toolbox’. Together, the development and implementation of a catalog of custom Cas editors and will accelerate and create a blueprint for the translation of safe and effective CRISPR therapies to benefit patients.