(a) Two monomers of FokI nuclease (red) fused to dCas9 (yellow) bind in complex with guide RNAs (sgRNA, green) to separate sites within the target locus. Only adjacently bound FokI-dCas9 monomers can assemble a catalytically active FokI nuclease dimer, triggering dsDNA cleavage. (b) FokI-dCas9 fusion architectures tested. Four distinct configurations of NLS, FokI nuclease, and dCas9 were assembled. 17 protein linker variants were also tested (see main text). (c) sgRNA orientation and (d) target sites tested within the EmGFP gene. Seven sgRNA target sites were chosen to test FokI-dCas9 activity in an orientation in which the PAM is distal from the cleaved spacer sequence (orientation A). Together, these seven sgRNAs enabled testing of FokI-dCas9 fusion variants across seven spacer lengths ranging from 5 to 43 bp. See for guide RNAs used to test orientation B, in which the PAM is adjacent to the spacer sequence.

Detection of genomic modification by loss of GFP signal or Surveyor assay at either an integrated GFP gene, or at endogenous genomic targets within the AAVS1, CLTA, EMX, HBB, or VEGF genes () (a) GFP disruption activity of fCas9, Cas9 nickase, or wild-type Cas9 with either no sgRNA, or sgRNA pairs of variable spacer length targeting the GFP gene in orientation A. (b) Indel modification efficiency from PAGE analysis of a Surveyor cleavage assay of renatured target-site DNA amplified from cells treated with fCas9, Cas9 nickase, or wild-type Cas9 and two sgRNAs spaced 14 bp apart targeting the GFP site (sgRNAs G3 and G7; ), each sgRNA individually, or no sgRNAs. The indel modification percentage is shown below each lane for samples with modification above the detection limit (~2%). (c-h) Indel modification efficiency for (c) two pairs of sgRNAs spaced 14 or 25 bp apart targeting the GFP site, (d) on pair of sgRNAs spaced 16 bp apart targeting the AAVS1 site, (e) one pair of sgRNAs spaced 19 bp apart targeting the CLTA site, (f) one pair of sgRNAs spaced 23 bp apart targeting the EMX site (g) one pair of sgRNAs spaced 16 bp apart targeting the HBB site, and (h) two pairs of sgRNAs spaced 14 or 16 bp apart targeting the VEGF site. Error bars reflect standard error of the mean from three biological replicates performed on different days.

(a) Results from high-throughput sequencing of GFP on-target sites amplified from 150 ng genomic DNA isolated from human cells treated with a plasmid expressing wild-type Cas9, Cas9 nickase, or fCas9; and either a plasmid expressing a single sgRNA (G1, G3, G5, or G7), or two plasmids each expressing a different sgRNA (G1+G5 or G3+G7). As a negative control, transfection and sequencing were performed in triplicate as above without any sgRNA expression plasmids. Sequences with more than one insertion or deletion at the GFP target site (the start of the G1 binding site to the end of the G7 binding site) were considered indels. Indel percentages are the number of indels observed divided by the total number of sequences. While wild-type Cas9 produced indels across all sgRNA treatments, fCas9 and Cas9 nickase produced indels efficiently (> 1%) only when paired sgRNAs were present. Indels induced by fCas9 and single sgRNAs were not detected at a frequency above that of the no-gRNA control, whereas Cas9 nickase and single sgRNAs modified the target GFP sequence at an average rate of 0.12%. (b-e) The indel mutation frequency from high-throughput DNA sequencing of amplified genomic on-target sites and off-target sites from human cells treated with fCas9, Cas9 nickase, or wild-type Cas9 and (b) two sgRNAs spaced 19 bp apart targeting the CLTA site (sgRNAs C1 and C2), (c) two sgRNAs spaced 23 bp apart targeting the EMX site (sgRNAs E1 and E2), or (d, e) two sgRNAs spaced 14 bp apart targeting the VEGF site (sgRNAs V1 and V2). (e) Two in-depth trials to measure genome modification at VEGF off-target site 1. Trial 1 used 150 ng of genomic input DNA and > 8 × 10⁵ sequence reads for each sample; trial 2 used 600 ng of genomic input DNA and > 23 × 10⁵ sequence reads for each sample. In (b-e), all significant (P value < 0.005 Fisher’s Exact Test) indel frequencies are shown. P values are listed in . For (b-e) each on- and off-target sample was sequenced once with > 10,000 sequences analyzed per on-target sample and an average of 76,260 sequences analyzed per off-target sample ().

(a-c) The indel mutation frequency from high-throughput DNA sequencing of amplified genomic on-target sites and off-target sites () from human cells treated with fCas9 or Cas9 nickase and (a) two sgRNAs (sgRNAs SA1 and SA2) spaced 15 bp apart targeting site A, human genomic locus chr1:21,655,401-21,655,461; (b) two sgRNAs (sgRNAs SB1 and SB2) spaced 24 bp apart targeting site B, human genomic locus chr2:31,485,447-31,485,516; or (c) two sgRNAs (sgRNAs SC1 and SC2) spaced 23 bp apart targeting the site C, human genomic locus chr3:48,747,484-48,747,552. P values are listed in . Each on- and off-target sample was sequenced once with > 10,000 sequences analyzed per on-target sample and an average of 83,000 sequences analyzed per off-target sample (). The mock transfection control represents the limit of detection for each site, determined from cells transfected with a GFP expression plasmid and no sgRNA or nuclease expression constructs.