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Nat Commun. 2017 Apr 7;8:14958. doi: 10.1038/ncomms14958.
Bacterial CRISPR-Cas systems comprise diverse effector endonucleases with different targeting ranges, specificities and enzymatic properties, but many of them are inactive in mammalian cells and are thus precluded from genome-editing applications. Here we show that the type II-B FnCas9 from Francisella novicida possesses novel properties, but its nuclease function is frequently inhibited at many genomic loci in living human cells. Moreover, we develop a proximal CRISPR (termed proxy-CRISPR) targeting method that restores FnCas9 nuclease activity in a target-specific manner. We further demonstrate that this proxy-CRISPR strategy is applicable to diverse CRISPR-Cas systems, including type II-C Cas9 and type V Cpf1 systems, and can facilitate precise gene editing even between identical genomic sites within the same genome. Our findings provide a novel strategy to enable use of diverse otherwise inactive CRISPR-Cas systems for genome-editing applications and a potential path to modulate the impact of chromatin microenvironments on genome modification.
细菌的 CRISPR-Cas 系统包含多种具有不同靶向范围、特异性和酶学特性的效应内切核酸酶，但其中许多在哺乳动物细胞中是无活性的，因此不能用于基因组编辑应用。在这里，我们表明来自弗朗西斯氏菌的 II-B 型 FnCas9 具有新的特性，但它的核酸酶功能在活的人类细胞中的许多基因组位点经常受到抑制。此外，我们开发了一种近端 CRISPR（称为代理-CRISPR）靶向方法，以特异性方式恢复 FnCas9 的核酸酶活性。我们进一步证明，这种代理-CRISPR 策略适用于多种 CRISPR-Cas 系统，包括 II-C 型 Cas9 和 V 型 Cpf1 系统，并且即使在同一基因组内相同的基因组位点之间也可以促进精确的基因编辑。我们的研究结果为利用多种其他无活性的 CRISPR-Cas 系统进行基因组编辑应用提供了一种新的策略，并为调节染色质微环境对基因组修饰的影响提供了一种潜在的途径。
