Programmable Live‐Cell CRISPR Imaging with Toehold‐Switch‐Mediated Strand Displacement. (7th September 2020)

• Record Type: Journal Article
• Authors: Hao, Yaya; Li, Jiang; Li, Qian; Zhang, Luhao; Shi, Jiye; Zhang, Xueli; Aldalbahi, Ali; Wang, Lihua; Fan, Chunhai; Wang, Fei
• Is Part Of: Angewandte Chemie. Volume 132:Number 46(2020)
• Page Start: 20793
• Page End: 20799
• Publication Date: 2020-09-07
• Subjects: cell imaging -- CRISPR-Cas9 -- DNA editing -- DNA hybridization -- strand-displacement reactions; Chemistry -- Periodicals; 540
• DOI: 10.1002/ange.202009062
• Languages: English
• ISSNs: 0044-8249

Abstract

Abstract: The widespread application of CRISPR‐Cas9 has transformed genome engineering. Nevertheless, the precision to control the targeting activity of Cas9 requires further improvement. We report a toehold‐switch‐based approach to engineer the conformation of single guide RNA (sgRNA) for programmable activation of Cas9. This activation circuit is responsive to multiple inputs and can regulate the conformation of the sgRNA through toehold‐switch‐mediated strand displacement. We demonstrate the orthogonal suppression and activation of Cas9 with orthogonal DNA inputs. Combination of toehold switches leads to a variety of intracellular Cas9 activation programs with simultaneous and orthogonal responses, through which multiple genome loci are displayed in different colors in a controllable manner. This approach provides a new route for programing CRISPR in living cells for genome imaging and engineering. Abstract : We constructed toehold switches on the sgRNA of CRISPR‐Cas9 that can respond to DNA single strands to regulate genome engineering. Various control circuits, including simultaneous, orthogonal, and logic gate‐controlled activation are demonstrated for tuning the display of multiple genome loci. This conformation‐based strand‐displacement‐mediated strategy provides a new route for programmable genome imaging and engineering.