Work

Dimeric CRISPR RNA-Guided FokI-dCas9 Nucleases Directed by Truncated gRNAs for Highly Specific Genome Editing

Year: 2015

Type: article

Abstract: 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.

Source: Human Gene Therapy

DOI: 10.1089/hum.2015.084

Authors: Nicolas Wyvekens, Ved V. Topkar, Cyd Khayter, J. Keith Joung, Shengdar Q. Tsai

Institutions: Massachusetts General Hospital, Center for Computational and Integrative Biology, Massachusetts General Hospital, Harvard University

Language: English

Topic: CRISPR and Genetic Engineering

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: green