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, 33 (2), 139-42

A split-Cas9 Architecture for Inducible Genome Editing and Transcription Modulation

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A split-Cas9 Architecture for Inducible Genome Editing and Transcription Modulation

Bernd Zetsche et al. Nat Biotechnol.

Figures

Figure 1
Figure 1. Generation and optimization of inducible split-Cas9
(a) Ribbon representation of Cas9. Triangles indicate split sites for split-4 (green) and split-5 (red) (b) Diagram of inducible split Cas9 fusions. N- and C-term pieces of human codon-optimized S.pyogenes Cas9 are fused to FRB and FKBP dimerization domains, respectively. (c and d) Strategy for optimizing the split Cas9 system. In the absence of rapamycin (c), the Cas9(N)-FRB-NES piece is sequestered in the cytoplasm due to the addition of an NES. The Cas9(C)-FKBP piece contains two NLSs and is actively imported into the nucleus. In the presence of rapamycin (d), Cas9(N)-FRB-NES binds to Cas9(C)-FKBP. NLSs of the resulting reassembled Cas9 mediate nuclear importation and subsequent binding to the targeted locus. (e) Representative SURVEYOR assay for split-4 and -5 mediated indels at the human EMX1 locus, with (left) and without (right) rapamycin. Arrowheads indicate expected SURVEYOR fragments. Nd = not detected (f) Schematic of lentiviral split Cas9 plasmid containing U6 promoter-driven sgRNA, EFS promoter-driven split Cas9 pieces and puromycin resistance gene (puro). 2A self-cleaving peptides (P2A) separate both split Cas9 pieces and puro. (g) Indel frequencies measured by deep sequencing at the EMX1 locus and four annotated OT. Indels were measured 4 weeks (wt-Cas9; n=2 biological replicates) or 6 weeks (split Cas9; n=3 biological replicates) after transduction (****p<0.0001). Rapamycin treatments lasted 12 days. Mean ± s.e.m. in all panels.
Figure 2
Figure 2. Inducible transcriptional activation using split dCas9-VP64 fusions
(a) Schematic of dCas9(N)-FRB-2xNES and dCas9(C)-FKBP-2xNLS-VP64 fusions used for transcriptional activation. Each piece harbors an annotated point mutation (D10A or N863A), which reconstitutes a dead Cas9 upon rapamycin-induced assembly. A VP64 transcriptional activator domain is fused to the C-term end of the dCas9(C)-FKBP-2xNLS-VP64 piece. (b) ASCL1 expression measured by qPCR in HEK293FT cells transfected with split-4 (Split) and four sgRNAs per gene. Expression was measured in cells with and without rapamycin (n=4 biological replicates), compared to full-length dead Cas9-VP64 (full-length) (n=3 biological replicates). Untransfected cells were used as baseline. (c) Neurog2 expression in N2A cells measured by qPCR 2, 6, 12, 24 and 72 hours after rapamycin treatment (n=3 biological replicates for each time point). Cells were treated continual with rapamycin (dark blue circles), only treated for 2 hours (light blue squares) or untreated (orange triangles). Untransfected cell were used as baseline. Mean ± s.e.m. in all panels.

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