Engineering CRISPR-Cas proteins for conditional and robust interrogation of the genome

项目摘要 Cas9是一种RNA引导的DNA靶向内切酶。它的可编程性促进了两个非常有用的 以前难以设计的功能：引入DNA双链或 通过融合，使蛋白质效应器断裂或重新聚集到基因组中所需的位置。作为一名 结果，Cas9彻底改变了基因编辑和基因组的功能询问。 尽管有这样的潜力，但一些问题限制了卡斯9的S的实用价值。其不受控制的性质限制了- 目标内切酶活性和促进最理想的基因组编辑形式的能力， 同源重组。与之相关的是，Cas9的S并不一定具有核酸内切酶的天然功能 与模块化融合蛋白支架一致，许多效应器融合未能引发可靠的 活动。因此，我们的工作目标是创造下一代CRISPR-Cas蛋白， 实现精确控制的基因组编辑活动和强大的融合蛋白活性。我们最近做了 开发了一系列围绕转座子介导的重组形成的工具，这些工具促进了 高效工程Cas9融合蛋白的构建及分离。使用这些工具，我们拥有 设计了一种变构调控的Cas9。在这里，使用这个和优化的变体，我们建议 研究CAS9的精确时间控制如何改善目标切割的预期活动 和同源重组。关于融合蛋白的构建，我们有初步的数据 这表明可以使用循环排列来改变Cas9的拓扑结构，原则上， 为效应器招募提供了一种新的蛋白质支架。我们建议系统地绘制 Cas9‘S在其初级序列上的循环排列势并使用这些非自然变体 为了改进一类非常有用的Cas9-效应因子，转录激活因子。最后，在这个过程中 在我们的初步实验中，我们偶然发现循环排列使得 门控Cas9蛋白的构建，该蛋白在特定的蛋白水解性切割时被激活。我们建议 开发这些分子作为一类新的基因组编辑工具，并在模型中演示它们的用途 植物中的病毒感染系统。如果成功，这些实验将解决几个主要的 今天基因组编辑界面临的挑战：如何减少偏离目标的影响，增加 同源重组活性，并利用Cas9-融合蛋白的可编程性 全速前进。

Project Summary Cas9 is an RNA-guided DNA-targeting endonuclease. Its programmability facilitates two extremely useful functionalities that were previously difficult to engineer: the introduction of either a DNA double strand break or recruitment, via fusion, of a protein effector to a desired location in the genome. As a consequence, Cas9 has revolutionized both gene editing and functional interrogation of the genome. Despite this potential, a number of issues constrain Cas9's utility. Its uncontrolled nature limits both on- target endonuclease activity and the ability to promote the most desirable form of genome editing, homologous recombination. Relatedly, Cas9's natural function as an endonuclease is not necessarily congruent with that of a modular fusion protein scaffold and many effector fusions fail to elicit reliable activity. The goal of our work is therefore to create the next-generation of CRISPR-Cas proteins that enable finely-controlled genome editing activity and robust fusion protein activity. We have recently developed a series of tools, fashioned around transposon-mediated recombination, that facilitate the construction and isolation of highly engineered Cas9 fusion proteins. Using these tools, we have engineered an allosterically regulated Cas9. Here, using this and optimized variants, we propose to investigate how precise temporal control of Cas9 can improve the desired activities of on-target cutting and homologous recombination. With regards to fusion protein construction, we have preliminary data suggesting that the topology of Cas9 can be altered using circular permutation which, in principle, provides a new class of protein scaffolds for effector recruitment. We propose to systematically map Cas9's potential for circular permutation across its primary sequence and use these non-natural variants to improve one class of highly useful Cas9-effectors, transcriptional activators. Finally, in the course of our preliminary experiments, we have serendipitously discovered that circular permutation allows the construction of gated Cas9 proteins that are activated upon specific proteolytic cleavage. We propose to develop these molecules as a new class of genome editing tool and demonstrate their utility in a model system of viral infection in planta. If successful, these experiments will address several of the major challenges facing the genome editing community today: how to reduce off-target effects, increase homologous recombination activity, and exploit the programmable nature of Cas9-fusion proteins to their full extent.