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Discovering the Genome-Wide Activity of CRISPR-Cas Nucleases.
ACS Chemical Biology 2018 Februrary 17
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.
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