Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing.

Shengdar Q. Tsai, Nicolas Wyvekens, Cyd Khayter, Jennifer A. Foden, Vishal Thapar, Deepak Reyon, Mathew J. Goodwin, Martin J. Aryee, J. Keith Joung
Source: Nat Biotechnol
Publication Date: (2014)
Issue: 32(6): 569-76
Research Area:
Molecular Biology
Cells used in publication:
U-2 OS
Species: human
Tissue Origin: bone
4D-Nucleofector® X-Unit
U2OS cells and U2OS.EGFP cells were transfected using the DN-100 program of a Lonza 4D-Nucleofector according to the manufacturer’s instructions. In Csy4 optimization experiments, 500 ng of pCAG-Csy4-T2A-Cas9 (pSQT834) and 250 ng of gRNA encoding plasmids were transfected with 50 ng of tdTomato expression plasmid (Clontech) as a transfection control. In initial FokI-dCas9 activity screens and focused spacer length analysis experiments, 750 ng of pCAG-Csy4-FokI-dCas9-nls nuclease plasmid (pJAF1484) and 250 ng of gRNA encoding plasmids were transfected together with 50 ng tdTomato expression plasmid as a transfection control. In all other experiments in U2OS and U2OS.EGFP cells, 975 ng of human codon optimized pCAG-hCsy4-T2A-nls-hFokI-dCas9-nls (pSQT1601) or pCAG-Csy4-T2A-Cas9-D10A nickase (pNW3) were transfected along with 325 ng of gRNA vector and 10 ng of tdTomato expression plasmid and analyzed 3 days after transfection. HEK293 cells were transfected with 750 ng of pSQT1601 nuclease plasmid, 250 ng of gRNA expression plasmid and 10 ng of Td tomato, using Lipofectamine (Life Technologies) according to the manufacturer’s instructions and analyzed for NHEJ-mediated mutagenesis 3 days after transfection. Single transfections were performed for the initial spacer activity screen, duplicate transfections for the focused spacer length analysis and quadruplicate transfections for the comparison of the activity of FokI-dCas9 with wildtype Cas9. All other transfections were performed in triplicate.
Monomeric CRISPR-Cas9 nucleases are widely used for targeted genome editing but can induce unwanted off-target mutations with high frequencies. Here we describe dimeric RNA-guided FokI nucleases (RFNs) that can recognize extended sequences and edit endogenous genes with high efficiencies in human cells. RFN cleavage activity depends strictly on the binding of two guide RNAs (gRNAs) to DNA with a defined spacing and orientation substantially reducing the likelihood that a suitable target site will occur more than once in the genome and therefore improving specificities relative to wild-type Cas9 monomers. RFNs guided by a single gRNA generally induce lower levels of unwanted mutations than matched monomeric Cas9 nickases. In addition, we describe a simple method for expressing multiple gRNAs bearing any 5' end nucleotide, which gives dimeric RFNs a broad targeting range. RFNs combine the ease of RNA-based targeting with the specificity enhancement inherent to dimerization and are likely to be useful in applications that require highly precise genome editing.