Research ArticleHEALTH AND MEDICINE

Engineering a far-red light–activated split-Cas9 system for remote-controlled genome editing of internal organs and tumors

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Science Advances  10 Jul 2020:
Vol. 6, no. 28, eabb1777
DOI: 10.1126/sciadv.abb1777
  • Fig. 1 Design of the FAST system.

    (A) Schematic of the split-Cas9 fusion protein components of the FAST system. Coh2 and DocS are two C. thermocellum proteins that interact with high affinity. Cas9 is formed from two separate (N- and C-terminal) Cas9 fragments that individually lack nuclease activity. When Cas9’s two fragments Cas9(N) and Cas9(C) are respectively fused with Coh2 and DocS, they readily combine to reconstitute a nuclease-active form of Cas9. (B) Schematic of the FAST system, as deployed in mammalian cells, based on the fragments detailed in (A). FRL (~730 nm) activates the engineered bacterial photoreceptor BphS, which converts guanosine triposphate (GTP) into c-di-GMP. c-di-GMP can bind to BldD (derived from sporulating actinomycete bacteria) and be translocated into the nucleus. This induces dimerization of the synthetic transcriptional activators p65-VP64-BldD [BldD fused with p65 (the nuclear factor κB–transactivating domain) and VP64 (a tetramer of the herpes simplex virus–derived VP16 activation domain)], after which they bind to PFRL to activate expression of the N-terminal fusion fragment of split-Cas9. The other (C-terminal) fusion fragment is constitutively expressed, as driven by the human cytomegalovirus promoter (PhCMV). DNA double-strand breaks are formed by Cas9 after the Coh2-DocS heterodimerization–mediated reconstitution of the two fusion fragments.

  • Fig. 2 Optimization of the FAST system.

    (A) Time schedule of FRL-controlled gene editing in HEK-293 cells. Cells were illuminated (1 mW/cm2; 730 nm) for 4 hours once a day for 2 days and were collected at 48 hours after the first illumination for further analysis. (B) A mismatch-sensitive T7 endonuclease I (T7E1) assay to test HEK-293 cells (6 × 104) transfected with full-length Cas9 (pHP1) or the FAST system (pXY137, pYH20, and pYH102), together with the sgRNA targeting to CCR5 locus (pYW57). FRL-mediated editing (indel deletions) of the human EMX1, CXCR4, and VEGFA loci by FAST was performed using the same experimental procedure as that used when targeting the CCR5 gene. (C) FRL-mediated multiplex editing of the human CCR5 and CXCR4 loci. (D) FAST-mediated DNA insertion via homology-directed repair (HDR), achieved by adding a single-stranded oligodeoxynucleotide (ssODN) template (10 μM), bearing a HindIII restriction endonuclease site. Homologous arms are indicated in red. The target sites of sgRNA (EMX1) are marked in blue. HEK-293 cells (6 × 104) were cotransfected with full-length Cas9 (pHP1) or the FAST system (pXY137, pYH20, and pYH102) and the sgRNA targeting to EMX1 locus (pYH227) via a nucleofection method. In (B) to (D), n = 2 from two independent experiments. Red arrows indicate the expected cleavage bands. Detailed description of genetic components and transfection mixtures are provided in tables S1 and S5. N.D., not detectable.

  • Fig. 3 Characterization of the FAST system.

    (A) FAST-mediated gene editing in four human cell lines. (B) Illumination intensity–dependent FAST gene editing. In (A) and (B), cells were collected for mismatch-sensitive T7E1 assays, as indicated in the time schedule of Fig. 2A. (C) Evaluation of exposure time–dependent FAST system gene editing performance. Cells were collected for T7E1 assays at 24 hours after the start of the second illumination. (D) Schematic of the photomask device used to demonstrate the spatial regulation of FAST-mediated gene editing. Cells were illuminated through a photomask containing a 7-mm line pattern. (E) Spatial control of FRL-dependent gene editing mediated by the FAST system. HEK-293 cells (3 × 106) were cotransfected with the FAST system, sgRNA (pYW57), and a frameshift enhanced green fluorescent protein (EGFP) reporter containing a CCR5 locus (pYH244) and were illuminated with FRL (0.5 mW/cm2; 730 nm; 2-min on, 2-min off) for 48 hours. EGFP is not expressed without Cas9 activity because the EGFP sequence is out of frame. Upon double-strand cleavage by Cas9, the frameshifts caused via DNA repair by NHEJ enable EGFP expression. The fluorescence of EGFP was assessed via fluorescence meter ChemiScope 4300 Pro imaging equipment (Clinx) at 48 hours. In (A) to (C), n = 2 from two independent experiments. Red arrows indicate the expected cleavage bands. Detailed description of genetic components and transfection mixtures are provided in tables S1 and S5. SEAP, human placental secreted alkaline phosphatase.

  • Fig. 4 FRL-induced FAST-mediated gene editing (CCR5 locus) of cells residing subcutaneously in mouse implants.

    (A) Schematic for the time schedule and experimental procedure for FRL-controlled gene editing in mice harboring hollow fiber implants with HEK-293 cells. Pairs of 2.5-cm hollow fibers containing a total of 5 × 106 transgenic HEK-293 cells (engineered with FAST system) were subcutaneously implanted on the dorsum of wild-type mice and illuminated with FRL (10 mW/cm2; 730 nm; 2-min on, 2-min off) for 4 hours each day for 2 days. Cells were collected from the hollow fiber implants at 48 hours after the first illumination and assessed with mismatch-sensitive T7E1 assay to assess targeted gene editing efficiency (CCR5 locus). (B) Representative T7E1 assay for FAST-mediated indel mutations. n = 3 mice. The red arrow indicates the expected cleavage bands. Detailed description of genetic components and transfection mixtures are provided in table S1 and S5.

  • Fig. 5 FAST-mediated gene editing of the tdTomato reporter locus in Gt(ROSA)26Sortm14(CAG-tdTomato)Hze mice.

    (A) Schematic showing the time schedule and experimental procedure for assessing in vivo gene editing. The minicircle iteration of the FAST system pYH412, pYH413, and pYH414 at a 7:15:4 (w/w/w) ratio were injected hydrodynamically via tail vein. Twenty-four hours after injection, mice were illuminated with FRL (10 mW/cm2; 730 nm; 2-min on, 2-min off) for 4 hours per day for 3 days. A second injection of the minicircle-based FAST system components was performed on the fifth day, followed by 4 hours daily illumination for three additional days. In our design, the tdTomato reporter protein was expressed after a stop cassette was destroyed by Cas9 editing. (B) Fluorescence IVIS image of mouse livers. (C) The frequency of edits (targeting the aforementioned stop cassette) by monitoring fluorescence intensity of the tdTomato reporter in Gt(ROSA)26Sortm14(CAG-tdTomato)Hze mice. (D) Representative fluorescence microscopy images of tdTomato and tdTomato+ hepatocytes present in frozen liver sections from FRL-illuminated mice. Blue indicates 4′,6-diamidino-2-phenylindole (DAPI) staining nuclei; red indicates endogenous tdTomato expression. The images represent typical results from three independent measurements. Scale bar, 100 μm. Data in (C) are means ± SEM; n = 3 mice. P values were calculated by Student’s t test. ****P < 0.0001 versus control.

  • Fig. 6 In-tumor gene editing (PLK1 locus) with the FAST system in xenograft (A549 cell) mice.

    (A) Schematic showing the time schedule and experimental procedure for the in-tumor FAST-mediated gene editing. The minicircle iteration of the FAST system targeting to PLK1 locus pYH412, pYH420, and pYH414 at a 7:15:4 (w/w/w) ratio were injected intratumorally. Twenty-four hours after per injection, mice were illuminated with FRL (10 mW/cm2; 730 nm; 2-min on, 2-min off) for 4 hours per day totally for 7 days. (B) Images of tumor tissues from the different treatments. (C) Tumor growth curves for the different treatments. (D) The weight of tumor tissues after the different treatments. (E) Indel mutations in the tumor tissues detected via mismatch-sensitive T7E1 assays. Red arrows indicate the expected cleavage bands. (F) The gene editing efficacy quantified by the TIDE analysis. (G) Relative mRNA expression levels of the PLK1 gene quantified by quantitative real-time polymerase chain reaction (qRT-PCR). The data are means ± SEM; n = 5 mice. P values were calculated by Student’s t test. ****P < 0.0001 versus control. (H) Representative fluorescence microscopy images of hematoxylin and eosin (H&E) staining of tumor tissues. The images represent typical results from three independent measurements. Scale bar, 100 μm. Representative fluorescence microscopy images of TUNEL staining (I) and caspase-3 (J) staining of tumor tissues. The images represent typical results from three independent measurements. Scale bars, 100 μm. Photo credit: Yuanhuan Yu, East China Normal University.

Supplementary Materials

  • Supplementary Materials

    Engineering a far-red light–activated split-Cas9 system for remote-controlled genome editing of internal organs and tumors

    Yuanhuan Yu, Xin Wu, Ningzi Guan, Jiawei Shao, Huiying Li, Yuxuan Chen, Yuan Ping, Dali Li, Haifeng Ye

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