Nucleic Acid-Based Anti-CRISPR Inhibitors of Cas9

项目总结 复杂、简便、有效的简单易用的Cas酶抑制剂仍然是主要的 缺少基于CRISPR的生物医学和治疗研究的工具。CRISPR-CAS的基本原理 抑制物包括需要更有效地产生模型细胞和生物体，威胁 在基于CRISPR的治疗中危险的偏离目标的编辑，以及对更精确控制的渴望 CAS酶活性在合成生物学和诊断中的应用。 已经开发了方法和改进的CaS酶来帮助解决这些问题和其他问题 CRISPR-Cas系统的缺点，特别是来自化脓性链球菌的Cas9(SpCas9)。这些 包括具有较低脱靶活性Cas9突变体和允许较小的 Cas9的分子或基于光的激活。然而，这些修改通常伴随着 减少目标上的活动，没有解决快速和具体关闭Cas9的需要。 尽管对CaS酶进行了改进，但对特定、广泛适用和易于使用的需求 抑制剂仍将是一种不可用的必要工具。可以抑制Cas9的小分子可能需要 开发的重大努力和潜在的负面影响，包括它们自己的脱离目标的影响和 对生物体微生物区系的无意影响，可能类似于抗生素的副作用。 最近发现的天然抗CRISPR蛋白可以与Cas9和其他Cas蛋白结合 亲和力很强。虽然它们很有希望作为抑制剂，但它们太大了，不能具有药物样的特性，而且它们的 最小化或优化并不是一个显而易见的练习。它们必须经过基因编码才能在细胞中使用 或者是有机体。然而，它们为设计一类新的CRISPR-Cas抑制剂提供了灵感。 在这里，我们建议开发一种新技术，基于核酸(NAB)的Cas9酶抑制剂。 这些分子比天然的抗CRISPR蛋白小，但可以以类似的亲和力结合， 化学合成，并很容易用普通方法导入细胞。在这个项目中，我们将 开发NAB抑制剂技术，优化其尺寸、化学、结合亲和力、抑制活性 和细胞稳定性。我们将进一步开发促进直接、无载体的方法和分子 NAB抑制剂的递送和定时释放。由此产生的NAB抑制剂预计将广泛地 适用和直接用于不同的生物医学研究。我们预计NAB抑制剂将 成为有价值的故障安全抑制剂，以克服基于CRISPR的疗法中的关键安全障碍。

PROJECT SUMMARY Sophisticated, facile, and potent inhibitors of Cas enzymes that are simple to use are still a major missing tool for CRISPR-based biomedical and therapeutic research. The rationale for CRISPR-Cas inhibitors includes a need for more efficient generation of model cells and organisms, the threat of dangerous off-target editing in CRISPR-based therapeutics, and the desire for more precise control over Cas enzyme activity in synthetic biology and diagnostics applications. Methods and modified Cas enzymes have been developed to help address these issues and other shortcomings of CRISPR-Cas systems, particularly Cas9 from Streptococcus pyogenes (SpCas9). These include Cas9 mutants with lower off-target activity and Cas9 mutants and protein fusions that allow small molecule or light-based activation of Cas9. However, these modifications typically have a concomitant decrease in on-target activity and do not address the need for rapid and specific shut-down of Cas9. Despite improvements to Cas enzymes, the need for specific, broadly applicable, and easy-to-use inhibitors will remain a necessary tool that is not available. Small molecules that can inhibit Cas9 may take significant effort to develop and potentially come with downsides, including their own off-target effects and inadvertent impacts on the organism’s microbiota, possibly similar to antibiotic side-effects. Natural anti-CRISPR proteins have recently been discovered that bind Cas9 and other Cas proteins with high affinity. While promising as inhibitors, they are too large to possess drug-like properties and their minimization or optimization is not an obvious exercise. They must be genetically encoded for use in cells or organisms. However, they provide inspiration for the design of a new class of CRISPR-Cas inhibitors. Here we propose to develop a new technology, nucleic acid-based (NAB) inhibitors of Cas9 enzymes. These molecules are smaller than natural anti-CRISPR proteins but can bind with similar affinity, be chemically synthesized, and be readily introduced into cells with common methods. In this project, we will develop NAB inhibitor technology by optimizing their size, chemistry, binding affinity, inhibitory activity, and cellular stability. We will further develop methods and molecules that facilitate direct, carrier-free delivery and timed-release of NAB inhibitors. The resulting NAB inhibitors are expected to be broadly applicable and straightforward to use for diverse biomedical research. We expect NAB inhibitors to become valuable fail-safe inhibitors to overcome the critical safety hurdles in CRISPR-based therapeutics.