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Discovering the Genome-Wide Activity of CRISPR-Cas Nucleases
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2017-12-27 00:00:00 , DOI: 10.1021/acschembio.7b00847
Shengdar Q. Tsai 1
Affiliation  

Originally discovered as part of an adaptive bacterial defense system against the invasion of foreign phages, programmable CRISPR-Cas nucleases have emerged as remarkable enzymes with transformative potential for both biological research and clinical application. CRISPR-Cas nucleases likely evolved in their natural context to tolerate imperfect specificity in order to recognize mutant bacteriophages. However, in the context of biological research and clinical applications, high specificity is generally preferred. For therapeutic applications in particular, it is important to carefully and empirically define the genome-wide activity of engineered nucleases, as hundreds of millions to billions of cells may be modified in a single therapeutic dose. Over the past several years, a number of both cell-based and in vitro sensitive and unbiased genome-scale methods to define CRISPR-Cas nuclease specificity have been developed. These methods will play important complementary roles in better understanding their global specificity profiles and identifying optimal nucleases for applications that demand high precision editing. Improving the sensitivity of mutation detection by next-generation sequencing, developing assays to define the functional consequences of unintended off-target activity nuclease activity, and understanding the consequences of individual human genetic variation on gene editing activity will be important areas for future research and development.

中文翻译:

发现CRISPR-Cas核酸酶的全基因组活性

可编程CRISPR-Cas核酸酶最初是作为针对外来噬菌体入侵的自适应细菌防御系统的一部分而发现的,已作为具有卓越转化潜力的杰出酶出现,可用于生物学研究和临床应用。CRISPR-Cas核酸酶可能在其自然环境中进化以耐受不完美的特异性,以便识别突变型噬菌体。然而,在生物学研究和临床应用的背景下,通常优选高特异性。特别是对于治疗应用,重要的是仔细地和经验地定义工程化核酸酶的全基因组活性,因为数亿至数十亿个细胞可以在单个治疗剂量中被修饰。在过去的几年中,一些两种细胞为基础,已经开发了定义CRISPR-Cas核酸酶特异性的体外敏感和无偏见的基因组规模方法。这些方法将在更好地了解它们的全局特异性概况以及为需要高精度编辑的应用程序确定最佳核酸酶方面起重要的补充作用。通过下一代测序提高突变检测的敏感性,开发测定方法以定义意外脱靶活性核酸酶活性的功能后果,以及了解个体人类遗传变异对基因编辑活性的后果将是未来研究和开发的重要领域。
更新日期:2017-12-27
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