Development of an intein-mediated split–Cas9 system for gene therapy

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Key findings

• Npu DnaE split-inteins reconstitute full-length SpCas9 from two separately expressed halves by protein trans-splicing, which ligates the SpCas9 halves with a peptide bond and leaves no scar. In split-SpCas9 version 1 (v1) the DnaE N-intein is fused to the C-terminus of SpCas9 residues 1–573 and the DnaE C-intein to the N-terminus of SpCas9 residues 574–1368, split between Glu573 and Cys574. In version 2 (v2) the split lies between Lys637 and Thr638, giving SpCas9 1–637 plus SpCas9 638–1368. The first residue of the C-terminal Cas9 half is Cys, Ser or Thr to support efficient splicing, and the split sites are surface-exposed.
• Both intein-split SpCas9 versions, v1 (split at Glu573/Cys574) and v2 (split at Lys637/Thr638), match wild-type SpCas9 nuclease activity at a genome-integrated traffic light reporter (TLR) in Neuro-2a cells 48 h after transient transfection. Only co-expression of both intein-fused halves with a U6-driven gRNA produces fluorescent cells, which report NHEJ (TagRFP) plus HDR (mVenus) events. Either intein-fused half alone with the gRNA, or SpCas9 without a gRNA, gives no detectable signal.
• Intein-split SpCas9 edits endogenous mouse loci in Neuro-2a cells at frequencies comparable to wild-type SpCas9 48 h after transient transfection. Indel frequencies for wild-type SpCas9 versus the split system are 23.1% versus 22.7% at Fus (T7 endonuclease I assay, split v1), 64.0% versus 48.1% at Rosa26 with gRNA Rosa26#1 (T7 endonuclease I assay), 50.2% versus 42.9% at Rosa26 with gRNA Rosa26#3 (XbaI RFLP), and 22.7% versus 24.9% at Rab38 with gRNA Rab38#2 (XcmI RFLP). Neither intein-fused half alone produces indels.
• Two rAAV vectors, each carrying one intein-fused SpCas9 half (pAAV_crTLR#1_Nv1 and pAAV_crTLR#1_Cv1, CBh promoter, bGH polyadenylation signal, U6-driven gRNA crTLR#1), reconstitute active SpCas9 in cells after co-transduction, so the combined coding sequence avoids the ~4.7–5 kb rAAV packaging limit. Co-transduction raises TLR reporter activity about two orders of magnitude above negative control in human AAVS1 TLR/+ HEK 293T cells and about one order of magnitude in mouse Neuro-2a TLR cells. Transduction with either rAAV alone is indistinguishable from the negative control. The reporter detects only about one third of events, so actual nuclease activity is higher.
• Double nicking with SpCas9 D10A nickase and two gRNAs (crTLR#1 and crTLR#3) favours HDR over NHEJ relative to wild-type SpCas9 at the Neuro-2a TLR reporter, with reduced NHEJ. Increased HDR makes the paired-nickase strategy attractive for gene repair.
• A split-intein version of SpCas9 D10A nickase (v1, split at Glu573/Cys574) retains nickase function. At the Neuro-2a TLR reporter with gRNAs crTLR#1 and crTLR#3, split-SpCas9 D10A shows the same HDR-to-NHEJ ratio as unsplit SpCas9 D10A, slightly lower overall activity than wild-type SpCas9, HDR comparable to wild-type SpCas9, and greatly reduced NHEJ.
• An HDR donor can be carried on the same plasmid as one half of the split-intein SpCas9 system, leaving about 0.9 kb of the ~5 kb dual-vector capacity free for additional sequence. In plasmid transfection of Neuro-2a TLR cells, an N-terminal-half rAAV plasmid (pAAV_crTLR#1_Donor_Nv1) carrying a Venus-repair donor with homology arms gives HDR comparable to wild-type SpCas9 and split SpCas9 supplied with a separate donor plasmid. A donor flanked by CRISPR recognition sites (pAAV_crTLR#1_CRISPR-Donor_Nv1) gives lower nuclease activity and reduced HDR, probably because SpCas9 cleaves the donor and destabilises it.

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.