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

项目摘要 Cas9是RNA引导的DNA靶向核酸内切酶。它的可编程性促进了两个非常有用的 以前难以工程化的功能：引入DNA双链， 通过融合将蛋白质效应物断裂或募集到基因组中的所需位置。作为 因此，Cas9彻底改变了基因编辑和基因组的功能询问。 尽管有这种潜力，但许多问题限制了Cas9的效用。它不受控制的性质限制了- 靶核酸内切酶活性和促进最理想形式的基因组编辑的能力， 同源重组相关地，Cas9作为内切核酸酶的天然功能不一定是必需的。 与模块化融合蛋白支架的功能一致，并且许多效应融合物不能引起可靠的 活动因此，我们工作的目标是创造下一代CRISPR-Cas蛋白， 能够实现精细控制的基因组编辑活性和稳健的融合蛋白活性。我们最近 开发了一系列工具，围绕转座子介导的重组， 高度工程化的Cas9融合蛋白的构建和分离。使用这些工具，我们 改造了一种变构调节的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靶向切割的期望活性 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Cas9的拓扑结构可以使用循环置换来改变，原则上， 提供了一类新的蛋白质支架用于效应子募集。我们建议系统地绘制 Cas9在其一级序列上循环排列的潜力，并使用这些非天然变体 以改进一类非常有用的Cas9效应子，转录激活子。最后，在 我们的初步实验，我们偶然发现，循环排列允许 构建在特异性蛋白水解切割时被激活的门控Cas9蛋白。我们建议 开发这些分子作为一类新的基因组编辑工具，并在模型中展示它们的实用性 植物体内病毒感染系统。如果成功，这些实验将解决几个主要问题 当今基因组编辑社区面临的挑战：如何减少脱靶效应，增加 同源重组活性，并利用Cas9-融合蛋白的可编程性质， 全范围。

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.