Bridge Helix of Cas9 Modulates Target DNA Cleavage and Mismatch Tolerance
Bridge Helix of Cas9 Modulates Target DNA Cleavage and Mismatch Tolerance
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DOI：
10.1021/acs.biochem.8b01241
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发表时间：
2019-04-09
期刊：
影响因子：
2.9
通讯作者：
Rajan, Rakhi
中科院分区：
文献类型：
--
作者：
Babu, Kesavan;Amrani, Nadia;Rajan, Rakhi
CRISPR-Cas系统是RNA引导的核酸酶，为细菌和古菌提供适应性免疫保护，使其免受基因组材料的入侵。CRISPR靶向机制的可编程性质使其能够适应为强大的基因组工程工具。Cas9是一种II型CRISPR效应蛋白，由于单引导RNA可以指导Cas9切割所需的基因组靶点，因此已被广泛用于基因编辑应用。对蛋白质不同结构域和引导RNA诱导的Cas9构象变化在选择目标DNA中的作用的了解一直并将继续使开发具有减少脱靶效应的Cas9变体成为可能。以前已经确定，Cas9中存在的一个富含精氨酸的桥螺旋(BH)是其活性的关键。在本研究中，我们发现在化脓性链球菌Cas9的BH环区域内的两个脯氨酸替换通过积累缺口产物和减少靶DNA线性化来削弱DNA切割活性。这反过来又在DNA靶向方面提供了更高的选择性。我们讨论了BH环对靶DNA识别的可能机制。
CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection for bacteria and archaea against intruding genomic materials. The programmable nature of CRISPR-targeting mechanisms has enabled their adaptation as powerful genome engineering tools. Cas9, a type II CRISPR effector protein, has been widely used for gene-editing applications owing to the fact that a single-guide RNA can direct Cas9 to cleave desired genomic targets. An understanding of the role of different domains of the protein and guide RNA-induced conformational changes of Cas9 in selecting target DNA has been and continues to enable development of Cas9 variants with reduced off-targeting effects. It has been previously established that an arginine-rich bridge helix (BH) present in Cas9 is critical for its activity. In the present study, we show that two proline substitutions within a loop region of the BH of Streptococcus pyogenes Cas9 impair the DNA cleavage activity by accumulating nicked products and reducing target DNA linearization. This in turn imparts a higher selectivity in DNA targeting. We discuss the probable mechanisms by which the BH-loop contributes to target DNA recognition.