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, 43 (13), 6450-8

Development of an Intein-Mediated split-Cas9 System for Gene Therapy

Affiliations

Development of an Intein-Mediated split-Cas9 System for Gene Therapy

Dong-Jiunn Jeffery Truong et al. Nucleic Acids Res.

Abstract

Using CRISPR/Cas9, it is possible to target virtually any gene in any organism. A major limitation to its application in gene therapy is the size of Cas9 (>4 kb), impeding its efficient delivery via recombinant adeno-associated virus (rAAV). Therefore, we developed a split-Cas9 system, bypassing the packaging limit using split-inteins. Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted. We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes. An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A. Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR). In addition, we explored the possibility of delivering the repair template accommodated on the same dual-plasmid system, by transient transfection, showing an efficient HDR. Most importantly, we revealed for the first time that intein-mediated split-Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV.

Figures

Figure 1.
Figure 1.
Using split-inteins for split–Cas9 reconstitution. (A) Upon split-intein reconstitution, the split-intein moieties splice themselves out and ligate the flanking N- and C-terminal SpCas9 halves (exteins) resulting in the recovery of active full-length SpCas9. (B) Split-SpCas9 version 1: DnaE N-intein (orange) is fused to the C-terminus of SpCas91-573(N-Cas9, red), whereas DnaE C-intein (green) is fused to N-terminus of SpCas9574-1368 (C-Cas9, blue). Split-SpCas9 version 2: DnaE N-intein is fused to the C-terminus of SpCas91-637, whereas DnaE C-intein is fused to N-terminus of SpCas9638-1368. (C) To measure nuclease activity, and to distinguish between HDR and NHEJ events, a traffic light reporter system was used. NHEJ events introduced by SpCas9, will often create indels that lead to frameshift mutations, resulting in the expression of TagRFP. HDR events will result in the repair of the truncated Venus by the repair template given (donor DNA; left homology arm: 0.4 kb, right homology arm: 1.4 kb) and the removal of the stop codon in the center of Venus leading to the expression of full-length Venus. Translated regions are denoted as lines under the schematic DNA with translation start site and stop codon. The box inset shows a detail of the target sequence recognized by the gRNAs crTLR#1 and crTLR#3. CAG: synthetic mammalian promoter CAG; bGH: bovine growth hormone polyadenylation site.
Figure 2.
Figure 2.
Testing split–Cas9 efficiency in Neuro-2a TLR cell lines. (A) Overview of the WT and split–Cas9 expression plasmids used (Cas9, N-Cas9_N-Intein_v1, C-Intein_C-Cas9_v1, N-Cas9_N-Intein_v2, C-Intein_C-Cas9_v2, gRNA crTLR#1/#2). To ensure a high expression; a strong synthetic mammalian promoter (CAG, green) and a bovine growth hormone (bGH, red) polyadenylation site was used. Cas9 cDNA is shown in orange, N-intein in dark brown and C-Intein in light brown. NLS (dark red): Nuclear localization signal. FLAG and HA tag are shown in light and dark grey respectively. For gRNA expression (turquoise), U6 promoter was chosen (dark green). (B) Results after FACS: only transfection with both N- and C-terminal parts of the split–intein–Cas9 system for version 1 (v1) and version 2 (v2) resulted in nuclease activity similar to wild-type SpCas9, represented in HDR or NHEJ events; transfection with only one moiety did not show any observable HDR or NHEJ events. Shown are means ± SD of three independent experiments. (C) The split-intein-Cas9 system v1 was used to target the fused in sarcoma (Fus) gene's second last exon. The respective segment was PCR amplified with the annotated primers for further analysis. T7 endonuclease I assay was performed after PCR on the samples to investigate the occurrence of NHEJ events. After the assay, the samples were analyzed with a Bioanalyzer. The appearance of a second band indicates the presence of indels resulting from NHEJ events. (D) Targeting of Rosa26 locus with the gRNA Rosa26#1. Only indels were detected when SpCas9 wild-type or both SpCas9 moieties were transfected. (E) Targeting of Rosa26 locus with the gRNA Rosa26#3. Indels were detected by RFLP analysis. The XbaI resistant product could be only observed when SpCas9 wild-type or both SpCas9 moieties were transfected. (F) Targeting of Rab38 locus with the gRNA Rab38#2. Indels were detected by RFLP analysis. The XcmI resistant product could be only observed when SpCas9 wild-type or both SpCas9 moieties were transfected. (G) Quantification of the nuclease activity in each target sequence. FU: fluorescence units, s: seconds.
Figure 3.
Figure 3.
Demonstration that the split-intein split-SpCas9 system can be delivered by rAAV. (A) Overview of the split–Cas9 rAAV constructs (pAAV_crTLR#1_Nv1, pAAV_crTLR#1_Cv1). To ensure a high expression; a strong synthetic mammalian promoter (CBh, green) and a bovine growth hormone (bGH, red) polyadenylation site was used. Split Cas9 cDNA is shown in orange, N-intein in dark brown and C-Intein in light brown. NLS (dark red): Nuclear localization signal. FLAG and HA tag are shown in light and dark grey, respectively. For gRNA expression (turquoise), U6 promoter was chosen (dark green). The inverted terminal repeats (ITR) are shown in light blue and light green. (B and C) Only nuclease activity was detectable when the two rAAV carrying the two moieties were added to the AAVS1 TLR/+ (b) or to the Neuro-2a TLR cells (c). In the experiments with only one of the moieties, the nuclease activity was indistinguishable from the negative control. Shown are means ± SD of three independent experiments.
Figure 4.
Figure 4.
Plasmid transfection experiment for comparison of SpCas9 wild-type and nickase with its respective split versions, and of donor DNA as separate plasmid or accommodated directly on the AAV plasmid. (A) Overview of the split–Cas9 plasmids used to express SpCas9, SpCas9D10A and its split version (Cas9, Cas9D10A, N-Cas9_N-Intein_v1, C-Intein_C-Cas9_v1, gRNA crTLR#1/#2. (B) rAAV plasmids used: without donor sequence (pAAV_crTLR#1_Nv1), carrying the donor DNA flanked by CRISPR sites (pAAV_crTLR#1_CRISPR-Donor_Nv1) or not flanked (pAAV_crTLR#1_Donor_Nv1), C-Cas9 expression plasmid (pAAV_crTLR#1_Cv1). CBh promoter is shown in green, bovine growth hormone (bGH, red). Split Cas9 cDNA is shown in orange, N-intein in dark brown and C-Intein in light brown. NLS (dark red): Nuclear localization signal. FLAG and HA tag are shown in light and dark grey, respectively. For gRNA expression (turquoise), U6 promoter was chosen (dark green). The inverted terminal repeats (ITR) are shown in light blue and light green. Donor DNA sequence (Donor, dark green) (C) The double nicking strategy, SpCas9D10A in combination with two gRNAs, showed a preference for HDR compared to wild-type. This effect was also observed with split-SpCas9D10A but with decreased activity. With the different DNA donor strategies no differences were observed, but with donor DNA flanked by CRISPR sites reduced HDR was observed. Shown are means ± SD of three independent experiments.

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References

    1. Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337:816–821. - PMC - PubMed
    1. Yin H., Xue W., Chen S., Bogorad R.L., Benedetti E., Grompe M., Koteliansky V., Sharp P.A., Jacks T., Anderson D.G. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 2014;32:551–553. - PMC - PubMed
    1. Ding Q., Strong A., Patel K.M., Ng S.L., Gosis B.S., Regan S.N., Cowan C.A., Rader D.J., Musunuru K. Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing. Circ. Res. 2014;115:488–492. - PMC - PubMed
    1. Ahi Y.S., Bangari D.S., Mittal S.K. Adenoviral vector immunity: its implications and circumvention strategies. Curr. Gene Ther. 2011;11:307–320. - PMC - PubMed
    1. Basner-Tschakarjan E., Mingozzi F. Cell-mediated immunity to AAV vectors, evolving concepts and potential solutions. Front. Immunol. 2014;5:350. - PMC - PubMed

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