Monomeric clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated 9 (Cas9) nucleases have been widely adopted for simple and robust targeted genome editing but also have the potential to induce high-frequency off-target mutations. In principle, two orthogonal strategies for reducing off-target cleavage, truncated guide RNAs (tru-gRNAs) and dimerization-dependent RNA-guided FokI-dCas9 nucleases (RFNs), could be combined as tru-RFNs to further improve genome editing specificity. Here we identify a robust tru-RFN architecture that shows high activity in human cancer cell lines and embryonic stem cells. Additionally, we demonstrate that tru-gRNAs reduce the undesirable mutagenic effects of monomeric FokI-dCas9. Tru-RFNs combine the advantages of two orthogonal strategies for improving the specificity of CRISPR/Cas nucleases and therefore provide a highly specific platform for performing genome editing.
CITATION STYLE
Wyvekens, N., Topkar, V. V., Khayter, C., Joung, J. K., & Tsai, S. Q. (2015). Dimeric CRISPR RNA-Guided FokI-dCas9 Nucleases Directed by Truncated gRNAs for Highly Specific Genome Editing. Human Gene Therapy, 26(7), 425–431. https://doi.org/10.1089/hum.2015.084
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