Deep mutational scanning is a powerful method for exploring the mutational fitness landscape of proteins. Its adaptation to anti-CRISPR proteins, which are natural CRISPR-Cas inhibitors and key players in the co-evolution of microbes and phages, facilitates their characterization and optimization. Here, we developed a robust anti-CRISPR deep mutational scanning pipeline in Escherichia coli that combines synthetic gene circuits based on CRISPR interference with flow cytometry coupled sequencing and mathematical modeling. Using this pipeline, we characterized comprehensive single point mutation libraries for AcrIIA4 and AcrIIA5, two potent inhibitors of CRISPR-Cas9. The resulting mutational fitness landscapes revealed considerable mutational tolerance for both Acrs, suggesting an intrinsic redundancy with respect to Cas9 inhibitory features, and – for AcrIIA5 – indicated mutations that boost Cas9 inhibition. Subsequent in vitro characterization suggested that the observed differences in inhibitory potency between mutant inhibitors were mostly due to changes in binding affinity rather than protein expression levels. Finally, to demonstrate that our pipeline can inform Acrs-based genome editing applications, we employed a selected subset of mutant inhibitors to increase CRISPR-Cas9 target specificity by modulating Cas9 activity. Taken together, our work establishes deep mutational scanning as a powerful method for anti-CRISPR protein characterization and optimization.

中文翻译：

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用于表征抗 CRISPR 蛋白适应度景观的深度突变扫描平台


深度突变扫描是探索蛋白质突变适应度景观的强大方法。它对抗 CRISPR 蛋白的适应，抗 CRISPR 蛋白是天然的 CRISPR-Cas 抑制剂，也是微生物和噬菌体共同进化的关键参与者，有助于它们的表征和优化。在这里，我们在大肠杆菌中开发了一种强大的抗 CRISPR 深度突变扫描管道，它将基于 CRISPR 干扰的合成基因电路与流式细胞术耦合测序和数学建模相结合。使用该管道，我们表征了 AcrIIA4 和 AcrIIA5 的综合单点突变文库，这两种有效的 CRISPR-Cas9 抑制剂。由此产生的突变适应度景观揭示了两种 Acrs 的相当大的突变耐受性，表明 Cas9 抑制特征存在内在冗余，并且 - 对于 AcrIIA5 - 表明增强 Cas9 抑制的突变。随后的体外表征表明，观察到的突变抑制剂之间抑制效力的差异主要是由于结合亲和力的变化，而不是蛋白质表达水平的变化。最后，为了证明我们的管道可以为基于 Acrs 的基因组编辑应用提供信息，我们采用了选定的突变抑制剂子集，通过调节 Cas9 活性来提高 CRISPR-Cas9 靶标特异性。综上所述，我们的工作将深度突变扫描确立为抗 CRISPR 蛋白质表征和优化的强大方法。