Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering

<div><p>The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15–39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.</p></div

fdCas9 exhibited significantly improved specificity.

<p>T7EI assays to determine the catalytic activity of fdCas9 on potential off-targets, identified by PROGNOS web-tool, for <i>EMX1</i>, <i>AAVS1</i>, <i>CCR5</i> and <i>HBB</i> genomic targets. T7EI mutation detection assays for potential off-targets of EMX1.4 and EMX1.14 gRNA pair (Fig 5A). T7EI mutation detection assays for potential off-targets of AAVS1.2 and AAVS1.10 gRNA pair (Fig 5B). T7EI mutation detection assays for potential off-targets of CCR5.13 and CCR5.15 gRNA pair (Fig 5C). T7EI mutation detection assays for potential off-targets of HBB9 and HBB10 gRNA pair (Fig 5D). Note: * indicates the expected size of the DNA bands of corresponding amplicons cleaved by T7EI.</p

Schematic representation of different dCas9 and FokI fusion variant architectures.

<p>(A) Schematic strategy used to test dCas9 and FokI fusion variants for homodimer formation and double-strand break (DSB) generation within the target sequence. A pair of gRNAs capable of guiding the dCas9 and FokI fusion variants and binding to the sense and antisense DNA strands to facilitate dimerization of the FokI catalytic domain is shown. (B) Schematic representation of dCas9 and FokI fusion variants. The FokI catalytic domain was fused to either the C- or N-terminus of dCas9 with different linker sequences to facilitate dimer formation. The fdCas9 variant was cloned under the CMV promoter with a linker of 16 amino acids and 4 NLSs, one in the N-terminal domain and three in the C-terminal domain. dCas9 was also cloned under the CMV promoter and used as a negative control. NLSs were included on either or both ends of the fusion protein to boost its nuclear localization.</p