DNA targeting by Clostridium cellulolyticum CRISPR-Cas9 Type II-C system
DNA targeting by Clostridium cellulolyticum CRISPR-Cas9 Type II-C system
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DOI：
10.1093/nar/gkz1225
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发表时间：
2020-02-28
影响因子：
14.9
通讯作者：
Severinov, Konstantin
中科院分区：
文献类型：
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作者：
Fedorova, Iana;Arseniev, Anatolii;Severinov, Konstantin
II型CRISPR-Cas9 RNA引导的核酸酶广泛用于基因组工程。来自化脓性链球菌的II-A型SpCas 9蛋白是其类别中研究最多且使用最多的酶。然而，它也有一些缺点，包括相对较大的尺寸、不完美的特异性和对侧接NGG PAM序列的DNA靶标的限制。来自其他细菌物种的Cas9直向同源物可以提供丰富且基本上未开发的生物化学多样性来源，这可以帮助克服SpCas 9的局限性。在这里，我们表征了CcCas 9，一种来自解纤维梭菌H10的II-C型CRISPR核酸酶。我们表明，CcCas 9是一种活性内切核酸酶，其大小相对较小，可识别一种新的两核苷酸PAM序列。CcCas 9可以潜在地拓宽Cas9核酸酶的生物技术应用的现有范围，并且对于C.纤维素分解菌H10，一种被认为是有前途的生物燃料生产者的细菌。
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.