Anti-CRISPR proteins (Acrs) block the activity of CRISPR-associated (Cas) proteins, either by inhibiting DNA interference or by preventing crRNA loading and complex formation. Although the main use of Acrs in genome engineering applications is to lower the cleavage activity of Cas proteins, they can also be instrumental for various other CRISPR-based applications. Here, we explore the genome editing potential of the thermoactive type II-C Cas9 variants from Geobacillus thermodenitrificans T12 (ThermoCas9) and Geobacillus stearothermophilus (GeoCas9) in Escherichia coli. We then demonstrate that the AcrIIC1 protein from Neisseria meningitidis robustly inhibits their DNA cleavage activity, but not their DNA binding capacity. Finally, we exploit these AcrIIC1:Cas9 complexes for gene silencing and base-editing, developing Acr base-editing tools. With these tools we pave the way for future engineering applications in mesophilic and thermophilic bacteria combining the activities of Acr and CRISPR-Cas proteins.

抗 CRISPR 蛋白 (Acrs) 通过抑制 DNA 干扰或阻止 crRNA 加载和复合物形成来阻断 CRISPR 相关 (Cas) 蛋白的活性。尽管 Acrs 在基因组工程应用中的主要用途是降低 Cas 蛋白的切割活性，但它们也可用于各种其他基于 CRISPR 的应用。在这里，我们探索了大肠杆菌中来自热脱氮地芽孢杆菌T12 (ThermoCas9) 和嗜热脂肪地芽孢杆菌(GeoCas9)的热活性 II-C Cas9 变体的基因组编辑潜力。然后我们证明来自脑膜炎奈瑟氏球菌的 AcrIIC1 蛋白强烈抑制其 DNA 切割活性，但不抑制其 DNA 结合能力。最后，我们利用这些 AcrIIC1:Cas9 复合物进行基因沉默和碱基编辑，开发 Acr 碱基编辑工具。借助这些工具，我们为未来在嗜温和嗜热细菌中结合 Acr 和 CRISPR-Cas 蛋白活性的工程应用铺平了道路。