A comparison of DNA repair pathways to achieve a site-specific gene modification of the Bruton's tyrosine kinase gene

Abstract

Gene editing utilizing homology-directed repair has advanced significantly for many monogenic diseases of the hematopoietic system in recent years but has also been hindered by decreases between in vitro and in vivo gene integration rates. Homology-directed repair occurs primarily in the S/G₂ phases of the cell cycle, whereas long-term engrafting hematopoietic stem cells are typically quiescent. Alternative methods for a targeted integration have been proposed including homology-independent targeted integration and precise integration into target chromosome, which utilize non-homologous end joining and microhomology-mediated end joining, respectively. Non-homologous end joining occurs throughout the cell cycle, while microhomology-mediated end joining occurs predominantly in the S phase. We compared these pathways for the integration of a corrective DNA cassette at the Bruton's tyrosine kinase gene for the treatment of X-linked agammaglobulinemia. Homology-directed repair generated the most integration in K562 cells; however, synchronizing cells into G₁ resulted in the highest integration rates with homology-independent targeted integration. Only homology-directed repair produced seamless junctions, making it optimal for targets where insertions and deletions are impermissible. Bulk CD34+ cells were best edited by homology-directed repair and precise integration into the target chromosome, while sorted hematopoietic stem cells contained similar integration rates using all corrective donors.

Keywords: CRISPR-Cas9; DNA repair pathways; HDR; HITI; PITCh; X-linked agammaglobulinemia; gene editing; homology-directed repair; homology-independent targeted integration; precise integration into target chromosome.

Graphical abstract

Figure 1

Editing schema and methods of targeted DNA integration (A) Diagram of proposed editing of the Bruton’s tyrosine kinase (BTK) gene. Boxes represent exons, while double lines are introns. Cas9/single-guide RNA (sgRNA) targeted to BTK intron 1 creates a double-stranded break (DSB) at the 3′ end of the intron. Simultaneous addition of an exogenous donor allows for one of the cell’s natural DSB repair pathways to be harnessed for targeted integration of the construct into the open-cut site. The integrated product will lead to RNA transcribed from the corrected BTK locus and production of functional protein. (B) DSBs can be repaired through multiple pathways, at least three of which can be harnessed for targeted integration of exogenous DNA. When end resection occurs, homology-directed repair (HDR) or PITCh can use homologous DNA sequences of varying lengths to guide integration into the DSB. These pathways occur predominantly in the S/G₂ and S phases of the cell cycle, respectively. HDR requires longer tracks of homology, while PITCh has short microhomology regions flanked by regions that will bind to the introduced ribonucleoprotein (RNP) and be cut. If no end resection occurs, homology-independent targeted integration (HITI) can be harnessed for integration. HITI donors have no homology to the cut site; however, they are flanked by 1 or 2 cut sites that match the RNP introduced, followed by end capture into the genomic locus. HITI can occur throughout all phases of the cell cycle. (C) Off-target analysis of the sgRNA targeting intron 1 of BTK as determined by genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq). (D) Schematics of the 9 donor variants evaluated. The red arrows together represent sgRNA/Cas9 binding sites with the rectangle representing the 17 bp of the protospacer on one side of the cut site, while the triangle represents the remaining 3 bp of protospacer and the protospacer adjacent motif (PAM). Two HDR donor variants were evaluated that differed only in the presence or absence of an intact PAM sequence. Four HITI donors were designed with sgRNA/Cas9 binding sites on both, either, or neither sides of the corrective donor template. Three PITCh donors were evaluated: two with sgRNA/Cas9 binding sites in differing orientations and one that lacked sgRNA sites entirely. In addition to the donor sequence, each donor contains 3 C-terminal hemagglutinin epitope tags to allow for the identification of transgenic protein products. (E) Diagram of the droplet digital PCR (ddPCR) amplicon used to measure integration rates of each donor template in K562 erythroleukemia cell lines. The reverse primer bound the codon-optimized sequence of the donor template, while the forward primer bound BTK intron 1 outside of the 5′ homology arm sequence found in any of the donors. Integration results from each condition are displayed when either the donor plasmid was electroporated into K562 cells alone (blue bars) or in conjunction with the sgRNA/Cas9 expression plasmid (red bars). Error bars represent standard deviation form the mean. (F) Representative immunoblot analysis of cells treated with both sgRNA/Cas9 and the respective donor template listed above. Lysates were probed for the hemagglutinin epitope tags and for an actin loading control.

Figure 2

Characterization of donor integration events in BTK deficient K562 cells BTK-deficient K562 cells were electroporated with the pX330 sgRNA/Cas9 expression plasmid and the corrective donor plasmid. The same primers used for ddPCR described in Figure 1D were used to characterize integration junctions using HDR ΔPAM, HDR, HITI-2c, and PITCh-2 donors. PCR products from each of the conditions were TOPO TA-cloned, and the resulting plasmids were Sanger-sequenced to identify the integration junctions produced by each DNA repair pathway. Sequencing results are shown in (A)–(D). The protospacer from each edit is highlighted in green, while the PAM sequence is in pink if it is intact or in red for the PAM-mutated donor. The sequence “TCCTCAG” is highlighted in red as the putative BTK intron 1 branchpoint. The cut site in the genomic DNA is represented by a vertical dotted line. In the HITI-1c5 donor, integration of the donor template protospacer was detected and is represented here in orange. The PITCh-2 microhomology arm is shown in blue. Inserted bases are shown as bolded text, while deleted bases are dashes. Finally, red boxes were used to illustrate micro-homologous DNA sequences flanking the cut site. (E) Summary of Sanger sequencing data.

Figure 3

Synchronization of K562 cells in G₁ increases corrective donor integration via HITI while decreasing integration from HDR and PITCh (A) Schematic of hydroxyurea (HU) synchronization. Cells were cultured with HU for 1 day before electroporation. Immediately preceding electroporation, cells underwent flow cytometric analysis for differences in cell cycle using a Hoechst stain. Following electroporation, cells were cultured for 3 days in media with or without HU, after which HU was removed. Cells were expanded in fresh media for 11 days before analysis for targeted integration via ddPCR. (B) Representative flow cytometry plots for untreated and HU-treated K562 cell populations on day 0. (C) Allelic disruption rates of K562 cells with or without HU culture. (D and E) Viability and fold expansion of cells 1-day post-electroporation were measured by trypan blue exclusion. (F) Targeted integration rates of corrective donors at the BTK locus in K562 cells. Gray bars represent cells grown without HU, while blue bars represent cells treated with HU (n = 4–8, 2–4 independent experiments). (G) Representative immunoblot from one of the experiments in Figure 4F detecting hemagglutinin tags (included at the 3′ end of each donor), which serves as a surrogate for expression of the integrated transgene. (H) In/Out PCR products from integration of HDR ΔPAM, HDR, HITI-1c5, and PITCh-2 donors into HU-treated K562 cells were TOPO TA-cloned, and the resulting plasmids were Sanger-sequenced to characterize integration junctions produced by each DNA donor. Data are presented as mean ± SD. Data in (C)–(F) were analyzed by Mann-Whitney test. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ns, not significant.

Figure 4

Synchronization of PBSCs in G₁ results in increased corrective donor integration via HITI while decreasing integration via the HDR pathway (A) Schematic of HU synchronization for peripheral blood stem cells (PBSCs). While in pre-stimulation media 1 day after thaw, 0.03 mg/mL HU was added to the culture. HU was maintained in the media until 1 day after gene modification, when both the AAV and HU were washed out. (B) Representative flow cytometry plots of PBSCs cultured with and without HU. (C and D) Viability (C) and fold expansion (D) of cells 1-day post-electroporation were measured by trypan blue exclusion. (E and F) Allelic disruption rates are shown in (E), while cDNA integration rates as measured by ddPCR are depicted in (F). Data are presented as mean ± SD. Data in (C)–(F) were analyzed by Mann-Whitney test. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ns, not significant.

Figure 5

Targeted integration rates in bulk and population-sorted primary human mobilized peripheral blood stem cells (A) Experimental timeline for gene editing of human mobilized PBSCs. (B) The percetange of allelic disruption as measured by Sanger sequencing (using inference of CRISPR edits [ICE]) in PBSCs using different combinations of sgRNA and Cas9. sgRNA was produced via in vitro transcription (IVT) or was chemically synthesized with 2′-O-methyl analogs and 3′ phosphorothioate internucleotide linkages at each of the three 5′ and 3′ terminal RNA residues (labeled “Mod”). Cas9 was delivered either as a protein pre-complexed with the gRNA (RNP) or as messenger RNA (n = 5–15 from 3 PBSC donor sources). (C) Targeted integration rates of each donor type in bulk CD34+ PBSCs. The three best-performing adeno-associated viral (AAV) donor vectors were tested for integration potential. (D) Representative flow cytometry plots demonstrating the sorting schema to differentiate between hematopoietic stem cells (HSCs: CD34+, CD38–, CD45RA–, CD90+) and progenitors (CD34+, CD38+). (E) Targeted integration rates of each donor type in bulk PBSCs (gray), progenitors (blue), or HSCs (red) as measured by ddPCR (n = 2–3 biological replicates from one of the PBSC donor sources from B and C). Cells were treated with chemically synthesized sgRNA pre-complexed to Cas9 as RNPs and AAV6 repair templates. Data are presented as mean ± SD. Data in (B), (C), and (E) were analyzed by Mann-Whitney test. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001, ns, not significant. For (E), p = 0.08 using the HDR donor and p = 0.2 using the PITCh donor.