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Protein Engineering Strategies to Expand CRISPR-Cas9 Applications

机译：扩展CRISPR-Cas9应用的蛋白质工程策略

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The development of precise and modulated methods for customized manipulation of DNA is an important objective for the study and engineering of biological processes and is essential for the optimization of gene therapy, metabolic flux, and synthetic gene networks. The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 is an RNA-guided site-specific DNA-binding complex that can be reprogrammed to specifically interact with a desired DNA sequence target. CRISPR-Cas9 has been used in a wide variety of applications ranging from basic science to the clinic, such as gene therapy, gene regulation, modifying epigenomes, and imaging chromosomes. Although Cas9 has been successfully used as a precise tool in all these applications, some limitations have also been reported, for instance (i) a strict dependence on a protospacer-adjacent motif (PAM) sequence, (ii) aberrant off-target activity, (iii) the large size of Cas9 is problematic for CRISPR delivery, and (iv) lack of modulation of protein binding and endonuclease activity, which is crucial for precise spatiotemporal control of gene expression or genome editing. These obstacles hinder the use of CRISPR for disease treatment and in wider biotechnological applications. Protein-engineering approaches offer solutions to overcome the limitations of Cas9 and generate robust and efficient tools for customized DNA manipulation. Here, recent protein-engineering approaches for expanding the versatility of the Streptococcus pyogenes Cas9 (SpCas9) is reviewed, with an emphasis on studies that improve or develop novel protein functions through domain fusion or splitting, rational design, and directed evolution.
机译：精确定制的DNA调控方法的开发对于生物学过程的研究和工程设计是重要的目标，对于优化基因疗法，代谢通量和合成基因网络至关重要。簇状规则间隔的短回文重复序列（CRISPR-）相关蛋白9是RNA引导的位点特异性DNA结合复合物，可以将其重新编程为与所需的DNA序列靶标特异性相互作用。 CRISPR-Cas9已在从基础科学到临床的广泛应用中使用，例如基因治疗，基因调节，修饰表观基因组和成像染色体。尽管Cas9已成功地在所有这些应用中用作精确工具，但也有一些局限性的报道，例如（i）严格依赖原间隔物相邻基序（PAM）序列，（ii）异常脱靶活性， （iii）Cas9的大尺寸对于CRISPR传递是有问题的，并且（iv）缺乏蛋白质结合和核酸内切酶活性的调节，这对于精确时空控制基因表达或基因组编辑至关重要。这些障碍阻碍了将CRISPR用于疾病治疗和更广泛的生物技术应用。蛋白质工程方法提供了解决方案，以克服Cas9的局限性，并为定制的DNA操作生成强大而有效的工具。在这里，综述了近年来用于扩展化脓链球菌Cas9（SpCas9）多功能性的蛋白质工程方法，重点是通过域融合或分裂，合理设计和定向进化来改善或开发新型蛋白质功能的研究。

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