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Hum Gene Ther. 2015 Jul;26(7):425-31. doi: 10.1089/hum.2015.084.

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

Author information

1
1 Molecular Pathology Unit & Center for Cancer Research, Massachusetts General Hospital , Charlestown, Massachusetts.
2
2 Center for Computational and Integrative Biology, Massachusetts General Hospital , Charlestown, Massachusetts.
3
3 Department of Pathology, Harvard Medical School, Boston, Massachusetts.

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.

PMID:
26068112
PMCID:
PMC4509490
DOI:
10.1089/hum.2015.084
[Indexed for MEDLINE]
Free PMC Article

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