Journal
JOURNAL OF BIOTECHNOLOGY
Volume 233, Issue -, Pages 74-83Publisher
ELSEVIER
DOI: 10.1016/j.jbiotec.2016.06.011
Keywords
RNA synthesis; Gene editing; Synthetic crRNA; Synthetic tracrRNA; Dual RNA
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Funding
- Dharmacon, part of GE Healthcare
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The CRISPR-Cas9 system has become the most popular and efficient method for genome engineering in mammalian cells. The Streptococcus pyogenes Cas9 nuclease can function with two types of guide RNAs: the native dual crRNA and tracrRNA (crRNA:tracrRNA) or a chimeric single guide RNA (sgRNA). Although sgRNAs expressed from a DNA vector are predominant in the literature, guide RNAs can be rapidly generated by chemical synthesis and provide equivalent functionality in gene editing experiments. This review highlights the attributes and advantages of chemically synthesized guide RNAs including the incorporation of chemical modifications to enhance gene editing efficiencies in certain applications. The use of synthetic guide RNAs is also uniquely suited to genome-scale high throughput arrayed screening, particularly when using complex phenotypic assays for functional genomics studies. Finally, the use of synthetic guide RNAs along with DNA-free sources of Cas9 (mRNA or protein) allows for transient CRISPR-Cas9 presence in the cell, thereby resulting in a decreased probability of off-target events. (C) 2016 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license.
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