title: Versatility of chemically synthesized guide RNAs for CRISPR-Cas9 genome editing.
abstract: 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.
authors.affiliation: Dharmacon, part of GE Healthcare, Lafayette, CO, 80026, USA.
authors.forename: Melissa L
authors.initials: ML
authors.lastname: Kelley
authors.affiliation: Dharmacon, part of GE Healthcare, Lafayette, CO, 80026, USA.
authors.forename: Žaklina
authors.initials: Ž
authors.lastname: Strezoska
authors.affiliation: Dharmacon, part of GE Healthcare, Lafayette, CO, 80026, USA.
authors.forename: Kaizhang
authors.initials: K
authors.lastname: He
authors.affiliation: Dharmacon, part of GE Healthcare, Lafayette, CO, 80026, USA.
authors.forename: Annaleen
authors.initials: A
authors.lastname: Vermeulen
authors.affiliation: Dharmacon, part of GE Healthcare, Lafayette, CO, 80026, USA. Electronic address: anja.smith@ge.com.
authors.forename: Anja van Brabant
authors.initials: Av
authors.lastname: Smith
mesh_terms: D064113:CRISPR-Cas Systems; D000072669:Gene Editing / Q000379:methods*; D000094704:RNA, Guide, CRISPR-Cas Systems; D013297:Streptococcus pyogenes / Q000235:genetics
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems
keywords: Dual RNA; Gene editing; RNA synthesis; Synthetic crRNA; Synthetic tracrRNA