2015/06/12 by Nicolas Wyvekens, Ved V. Topkar, Cyd Khayter +2 · 1 citation
Biochemistry, Genetics and Molecular Biology · #CRISPR and Genetic Engineering #RNA regulation and disease #RNA Interference and Gene Delivery
paper · doi:10.1089/hum.2015.084
openalex publication_date 2015/06/12 · openalex created_date 2025/10/10 · openalex updated_date 2026/08/01
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.