Dimeric CRISPR RNA-guided FokI Nuclease (RFN) Technology

This page lists plasmids for practicing the dimeric CRISPR RNA-guided FokI Nuclease ( RFN ) technology developed by the Joung lab (Tsai et al., Nat Biotechnol. 2014).

Use of dimeric RFNs requires the expression of two guide RNAs (gRNAs) and a FokI-dCas9 fusion protein. The pSQT1313 plasmid enables the expression of two gRNAs in a single transcript whose expression is driven by a U6 promoter. The two gRNAs expressed are flanked by Csy4 recognition sequences so that they can be cleaved out from the larger transcript by the site-specific Csy4 ribonuclease. Vectors expressing gRNAs targeted to sequences of interest can be made by cloning customized oligonucleotides into pSQT1313 using the protocol described in Supplementary Figure 8 of Tsai et al., Nat Biotechnol. 2014. The ZiFiT Targeter program from the Joung lab can be used both to identify potential target sites for RFNs and also provides the sequences of oligonucleotides to be synthesized for making specific gRNA pairs. ZiFiT Targeter is freely available online without registration.

pSQT1601 encodes the Csy4 ribonuclease (required for processing of gRNAs co-expressed in vectors made from pSQT1313) and the FokI-dCas9 protein as a fusion protein in which the two components are joined by a T2A self-cleaving peptide sequence. The Csy4 and FokI domains have been codon optimized for expression in human cells and the dCas9 domain has been optimized for expression in zebrafish and human cells. Expression of this fusion is driven by a CAG promoter. Note that the FokI-dCas9 fusion encoded by this plasmid is the one that showed optimal activity in human cells as described in Tsai et al., Nat Biotechnol. 2014.

Individual plasmids can be ordered via the links below:

ID	Plasmid
53370	pSQT1313 (multiplex gRNA expression plasmid)
53369	pSQT1601 (optimized Csy4 and FokI-dCas9 expression vector)