Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing.
Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin MJ, Aryee MJ, Joung JK
Nat Biotechnol. 2014 Apr 25. doi: 10.1038/nbt.2908.
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PubMed
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Article
Dimeric CRISPR RNA-guided FokI Nuclease (RFN) Technology
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 (Link opens in a new window) 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.
Plasmids from Article
| ID | Plasmid | Purpose |
|---|---|---|
| 53369 | pSQT1601 | human codon optimized Csy4 and FokI-dCas9 expression plasmid |
| 53370 | pSQT1313 | multiplex gRNA expression plasmid |
| 53371 | pSQT834 | Csy4 and Cas9 nuclease expression plasmid |
| 53372 | pNW3 | Csy4 and Cas9 D10A nickase expression plasmid |
| 53373 | pSQT817 | wildtype Cas9 expression plasmid |
| 60071 | RFN_EGFP_47 | Expresses a pair of control multiplex gRNAs targeting EGFP for use with dimeric hFokI-dCas9 nuclease |
| 60072 | RFN_EGFP_80 | Expresses a pair of control multiplex gRNAs targeting EGFP for use with dimeric hFokI-dCas9 nuclease |
| 60073 | RFN_EGFP_81 | Expresses a pair of control multiplex gRNAs targeting EGFP for use with dimeric hFokI-dCas9 nuclease |
| 60074 | RFN_EGFP_82 | Expresses a pair of control multiplex gRNAs targeting EGFP for use with dimeric hFokI-dCas9 nuclease |
| 60075 | RFN_EGFP_83 | Expresses a pair of control multiplex gRNAs targeting EGFP for use with dimeric hFokI-dCas9 nuclease |
| 60076 | RFN_EGFP_84 | Expresses a pair of control multiplex gRNAs targeting EGFP for use with dimeric hFokI-dCas9 nuclease |