Scalable Development of Custom Genome Editing Technologies

项目摘要 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 领域的尽管CRISPR-Cas酶用于基因组编辑的适应性已经促进和显著地改善了基因组编辑的效率。 加速了在活细胞中编辑核酸序列的能力，许多基因工程方法被 使用具有显著局限性的单一酶进行。天然存在的CRISPR-Cas9效应子 来自细菌酿脓链球菌（SpCas 9）的基因可以有效地用于某些编辑应用，但是 并不是一个“一刀切”的解决方案，用于治疗导致遗传疾病的序列的多样性。临床 SpCas 9的潜力由于酶的天然特性而固有地受到限制，包括要求 结合一个短的DNA基序来启动编辑。该基序仅发生在基因组的一小部分中，阻止了SpCas 9 编辑许多致病突变，而这些突变并不包含这个序列。SpCas 9无法靶向 广泛的DNA位点，沿着与其他次优特征，说明需要创新， 释放基因组编辑在临床上的巨大潜力。一个解决方案，使更全面的基因组 靶向是利用定向进化来设计可以识别新基序的SpCas 9的新形式。 然而，传统的蛋白质工程方法仍然是低通量的，昂贵的，费力的。这里我们将 通过优化可扩展的方法来缩小这些技术和方法上的差距， 具有改进性质的新Cas变体。我们提出的研究将解决突出的局限性， CRISPR-Cas酶通过：（1）开发可扩展的实验方法，以更快速有效地 工程和表征蛋白质，（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 对抗导致常见和罕见疾病的突变。该项目的长期愿景是创造一个 定制编辑器的目录，共同可以系统地靶向基因组，而不牺牲其他重要的 特性如特异性。为了使编辑完全民主化，这一系列优化的CRISPR技术将 为研究人员和临床医生创建一个虚拟的“一站式”，为患者寻求优化的基因治疗。 成功完成拟议的研究将协同实验和计算方法，将 为表征和改善基因组编辑的活性提供了新的可扩展的方法 技术，并将成倍扩大编辑'工具箱'内的功能。统称 开发和实施定制Cas编辑器目录，并将加速和创建蓝图 将安全有效的CRISPR疗法转化为有益于患者的疗法。

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.