Type II CRISPR-Cas9 RNA-guided nucleases are widely used for genome engineering. Type II-A SpCas9 protein from Streptococcus pyogenes is the most investigated and highly used enzyme of its class. Nevertheless, it has some drawbacks, including a relatively big size, imperfect specificity and restriction to DNA targets flanked by an NGG PAM sequence. Cas9 orthologs from other bacterial species may provide a rich and largely untapped source of biochemical diversity, which can help to overcome the limitations of SpCas9. Here, we characterize CcCas9, a Type II-C CRISPR nuclease from Clostridium cellulolyticum H10. We show that CcCas9 is an active endonuclease of comparatively small size that recognizes a novel two-nucleotide PAM sequence. The CcCas9 can potentially broaden the existing scope of biotechnological applications of Cas9 nucleases and may be particularly advantageous for genome editing of C. cellulolyticum H10, a bacterium considered to be a promising biofuel producer.

中文翻译：

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通过解纤梭菌CRISPR-Cas9 II-C型系统进行DNA靶向。

II型CRISPR-Cas9 RNA引导的核酸酶广泛用于基因组工程。化脓性链球菌的II-A型SpCas9蛋白是同类研究中使用最多的酶。然而，它具有一些缺点，包括相对较大的尺寸，不完善的特异性以及对与NGG PAM序列侧翼的DNA靶标的限制。来自其他细菌物种的Cas9直系同源物可能提供了丰富且尚未开发的生化多样性来源，这可以帮助克服SpCas9的局限性。在这里，我们表征了CcCas9，一种来自解纤梭菌H10的II-C型CRISPR核酸酶。我们显示，CcCas9是一种相对较小的活性核酸内切酶，可识别新型的两个核苷酸PAM序列。