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, 20 (3), 750-756

CRISPR-Mediated Integration of Large Gene Cassettes Using AAV Donor Vectors

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CRISPR-Mediated Integration of Large Gene Cassettes Using AAV Donor Vectors

Rasmus O Bak et al. Cell Rep.

Abstract

The CRISPR/Cas9 system has recently been shown to facilitate high levels of precise genome editing using adeno-associated viral (AAV) vectors to serve as donor template DNA during homologous recombination (HR). However, the maximum AAV packaging capacity of ∼4.5 kb limits the donor size. Here, we overcome this constraint by showing that two co-transduced AAV vectors can serve as donors during consecutive HR events for the integration of large transgenes. Importantly, the method involves a single-step procedure applicable to primary cells with relevance to therapeutic genome editing. We use the methodology in primary human T cells and CD34+ hematopoietic stem and progenitor cells to site-specifically integrate an expression cassette that, as a single donor vector, would otherwise amount to a total of 6.5 kb. This approach now provides an efficient way to integrate large transgene cassettes into the genomes of primary human cells using HR-mediated genome editing with AAV vectors.

Keywords: AAV; CRISPR/Cas9; gene editing; genome; integration; large; long; sequential.

Figures

Figure 1
Figure 1. Sequential homologous recombination of two AAV6 donors with a split gene
Schematic overview of a two-step HR platform, in which a gene is split between two HR donors (Donor A and B), which undergo sequential HR. Donor A carries a sgRNA target site (red box) immediately after ‘part A’ of the transgene. This allows HR of Donor B using the same sgRNA, which seamlessly fuses ‘part B’ of the transgene to ‘part A’. Stuffer DNA (white box) after the sgRNA target site is used as homology arm for Donor B to avoid re-using the right homology arm from Donor A.
Figure 2
Figure 2. Sequential HR targeting a GFP gene split between two AAV donor vectors to the CCR5 locus in K562 cells
(a) Overview of the donor design for splitting GFP between two AAV donors. The endogenous CCR5 target site is shown with the PAM in red and the 20nt target site in purple. The Cas9 cut site is between nucleotide 17 and 18 of the target sequence. Donor A is designed with 2 x 400 bp homology arms (LHA and RHA) that are split at the CCR5 cut site. The homology arms flank a PGK-BFP expression cassette, part A of the GFP expression cassette (SFFV-GFP (A)), a sgRNA target site for the same CCR5 sgRNA, and stuffer DNA (to serve as homology arm for Donor B to avoid having to re-use the 400bp CCR5 left homology arm). After HR of Donor A, Donor B is designed to seamlessly integrate the rest of GFP using the sgRNA target site present in Donor A. Donor B has a LHA homologous to GFP (begins at amino acid 57 of GFP), a RHA consisting of part of the sgRNA target site and the stuffer DNA, and it carries an EF1a-mCherry expression cassette. Neither donor expresses GFP on its own (Figure S1a and S1b). (b) GFP is split at a PAM site for the CCR5 sgRNA. Codons are depicted above the nucleotides. Donor A carries LHA and RHA which are split directly at the Cas9 cut site in CCR5 as depicted in (a). Donor A carries a truncated GFP sequence that ends after the PAM site identified in the GFP gene. Directly after the PAM, the 20nt target site for the same CCR5 sgRNA is introduced. Note that the last codon (Pro) of the truncated GFP sequence is maintained with the fusion to the sgRNA target sequence. Thus, the LHA of Donor B ends right after this proline codon. The right homology arm begins immediately after the Cas9 cut site (scissors). The two homology arms flank the remaining part of GFP and an mCherry expression cassette, see (a), that upon seamless HR of Donor B will reconstitute a functional GFP open reading frame. (c) K562 cells were mock-electroporated or electroporated with Cas9 mRNA and CCR5 synthetic sgRNAs (CRISPR) followed by transduction with the split GFP AAV6 donor pair. GFP expression was measured either by total percent GFP+ cells after 16 days or percent GFPhigh cells 8 days after transduction (see also Figure S2a). Left panel, representative FACS plots. Right panel, frequencies of cells stably expressing GFP, N = 7, error bars represent SD.
Figure 3
Figure 3. Sequential homologous recombination of two AAV6 donors with a split GFP gene in human T cells and CD34+ hematopoietic stem and progenitor cells
(a) Primary human T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs) were mock-electroporated or electroporated with Cas9 protein precomplexed with CCR5 chemically modified sgRNAs (CRISPR) followed by transduction with a split GFP AAV6 donor pair (Figure 2a). GFP expression was measured by flow cytometry four days after transduction. Left panel, representative FACS plots from the two cell types. Right panel, frequencies of GFP+ cells for the two cell types, N = 11 (T cells, all from different buffy coat donors), N = 12 (HSPCs, all from different cord blood donors). (b) HSPCs were treated as in (a) and at day 4 post-transduction, GFP+ cells were single-cell sorted into 96-well plates containing methylcellulose and progenitor-derived clones were visualized 14 days after seeding. Top panel, fluorescent microscopy images of formed GFP+ colonies from erythroid (BFU-E), granulocyte/macrophage (CFU-GM), and multi-lineage (CFU-GEMM) progenitors (scale bars: blue=200μm, red=1000μm, green=400μm). Bottom panel, In-Out PCR was performed on colony-derived genomic DNA to confirm targeted integration at the 5’ end (Donor A) and at the 3’ end (Donor B) (Figure S3i). Representative gel image of 6 clones of a total of 41 clones analyzed. Input control is PCR amplification of a part of the HBB gene. (c) HSPCs were treated as in (a) and at day 4 post-transduction, GFP+ cells were isolated by FACS and 40,000 cells were transplanted into each of three male NSG mice sublethally irradiated 24 hrs previously. Engraftment of human cells in the bone marrow was assessed 16 weeks post-transplant by flow cytometric detection of HLA-ABC/hCD45 double-positive cells and GFP expression was analyzed within the human population. FACS plots show a negative control mouse not transplanted with human cells and a representative FASC plot from one of the mice transplanted with GFP+ HSPCs (CRISPR + donors), N = 3.
Figure 4
Figure 4. Sequential homologous recombination of two AAV6 donors with a split EGFR gene in human T cells and CD34+ hematopoietic stem and progenitor cells
(a) Schematic overview of a two-step HR platform integrating an EGFR expression cassette into the CCR5 gene. Donor A carries all elements of the expression cassette, but only ‘part A’ of the EGFR coding sequence followed by the same sgRNA target site (red box) used for HR of Donor A. ‘Part B’ is introduced by HR using this sgRNA target site and is fused seamlessly with ‘part A’ thereby constituting a full EGFR open reading frame. (b) Primary human T cells and CD34+ HSPCs were mock-electroporated or electroporated with Cas9 protein precomplexed with CCR5 sgRNA (CRISPR) followed by transduction with the split EGFR AAV6 donor pair. Left panel, representative FASC plots showing EGFR expression four days post-transduction. Right panel, frequencies of EGFR+ cells measured four days post-transduction, N = 14 (T cells, all from different buffy coat donors), N = 9 (HSPCs, all from different cord blood donors). (c) HSPCs were treated as in (b) and at day 4 post-transduction, EGFR+ cells were single-cell sorted into 96-well plates containing methylcellulose and In-Out PCR was performed on genomic DNA from progenitor-derived clones 14 days after seeding. Representative gel image shows targeted integration of Donor A and B, confirmed by the 5’ end and 3’ end PCR, respectively, in 6 out of 20 total colonies. Input control is PCR amplification of part of the HBB gene.

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