Proteins evolve through the modular rearrangement of elements known as domains. It is hypothesized that extant, multidomain proteins are the result of domain accretion, but there has been limited experimental validation of this idea. Here, we introduce a technique for genetic minimization by iterative size-exclusion and recombination (MISER) that comprehensively assays all possible deletions of a protein. Using MISER, we generated a deletion landscape for the CRISPR protein Cas9. We found that Cas9 can tolerate large single deletions to the REC2, REC3, HNH, and RuvC domains, while still functioning in vitro and in vivo, and that these deletions can be stacked together to engineer minimal, DNA-binding effector proteins. In total, our results demonstrate that extant proteins retain significant modularity from the accretion process and, as genetic size is a major limitation for viral delivery systems, establish a general technique to improve genome editing and gene therapy-based therapeutics.

中文翻译：

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CRISPR-Cas9的全面缺失图谱可识别最小的RNA指导的DNA结合模块

蛋白质通过称为域的元件的模块重排而进化。据推测，现存的多结构域蛋白是结构域积聚的结果，但是对该想法的实验验证有限。在这里，我们介绍了一种通过迭代大小排除和重组（MISER）进行遗传最小化的技术，该技术可以全面检测蛋白质的所有可能缺失。使用MISER，我们为CRISPR蛋白Cas9生成了一个缺失态。我们发现Cas9可以耐受REC2，REC3，HNH和RuvC结构域的大的单个缺失，同时仍在体外和体内起作用，并且这些缺失可以堆叠在一起以工程化最小的DNA结合效应蛋白。总体而言，我们的结果表明，现存的蛋白质在吸积过程中保留了显着的模块化，并且