Optimization of scarless human stem cell genome editing

Abstract

Efficient strategies for precise genome editing in human-induced pluripotent cells (hiPSCs) will enable sophisticated genome engineering for research and clinical purposes. The development of programmable sequence-specific nucleases such as Transcription Activator-Like Effectors Nucleases (TALENs) and Cas9-gRNA allows genetic modifications to be made more efficiently at targeted sites of interest. However, many opportunities remain to optimize these tools and to enlarge their spheres of application. We present several improvements: First, we developed functional re-coded TALEs (reTALEs), which not only enable simple one-pot TALE synthesis but also allow TALE-based applications to be performed using lentiviral vectors. We then compared genome-editing efficiencies in hiPSCs mediated by 15 pairs of reTALENs and Cas9-gRNA targeting CCR5 and optimized ssODN design in conjunction with both methods for introducing specific mutations. We found Cas9-gRNA achieved 7-8× higher non-homologous end joining efficiencies (3%) than reTALENs (0.4%) and moderately superior homology-directed repair efficiencies (1.0 versus 0.6%) when combined with ssODN donors in hiPSCs. Using the optimal design, we demonstrated a streamlined process to generated seamlessly genome corrected hiPSCs within 3 weeks.

Figure 1.

Functional tests of re-TALENs in human somatic and stem cells. (a) Schematic representation of experimental design for testing genome targeting efficiency. A genomically integrated GFP-coding sequence is disrupted by the insertion of a stop codon and a 68 bp genomic fragment derived from the AAVS1 locus (bottom). Restoration of the GFP sequence by nuclease-mediated homologous recombination with tGFP donor (top) results in GFP+ cells that can be quantitated by FACS. Re-TALENs and TALENs target identical sequences within AAVS1 fragments. (b) Bar graph depicting GFP+ cell percentage introduced by tGFP donor alone, TALENs with tGFP donor and re-TALENs with tGFP donor at the target locus, as measured by FACS (N = 3, error bar = SD). Representative FACS plots are shown later in the text. (c) Schematic overview depicting the targeting strategy for the native AAVS1 locus. The donor plasmid, containing splicing acceptor (SA)- 2 A (self-cleaving peptides), puromycin resistant gene (PURO) and GFP were described before (14). The locations of PCR primers used to detect successful editing events are depicted as blue arrows. (d) Successfully targeted clones of PGP1 hiPSCs were selected with puromycin (0.5 µg/ml) for 2 weeks. Microscopy images of three representative GFP+ clones are shown. Cells were also stained for the pluripotency markers TRA-1-60. Scale bar: 200 µm. (e) PCR assays performed on these the monoclonal GFP+ hiPSC clones demonstrated successful insertions of the donor cassettes at the AAVS1 site (lanes 1–3), whereas plain hiPSCs show no evidence of successful insertion (lane C). (f) Sanger sequencing of the PCR amplicon from the three targeted hiPSC colonies confirmed that the expected DNA bases at the genome-insertion boundary is present.

Figure 2.

Comparison of reTALENs and Cas9-gRNAs genome targeting efficiency on CCR5 in iPSCs. (a) Schematic representation of genome engineering experimental design. At the re-TALEN pair or Cas9-gRNA targeting site, a 90 mer ssODN carrying a 2 bp mismatch against the genomic DNA was delivered along with the reTALEN or Cas9-gRNA constructs into PGP1 hiPSCs. The cutting sites of the nucleases are depicted as red arrows in the figure. (b) Deep-sequencing analysis of HDR and NHEJ efficiencies for re-TALEN pairs (CCR5 #3) and ssODN, or the Cas9-gRNA and ssODN. Alterations in the genome of hiPSCs were analyzed from high-throughput sequence data by GEAS. Top: HDR was quantified from the fraction of reads that contained a 2 bp point mutation built into the center of the ssODN (blue), and NHEJ activity was quantified from the fraction of deletions (gray)/Insertions (red) at each specific position in the genome. For the reTALEN and ssODN graphs, we plot green dashed lines to mark the outer boundary of the re-TALEN pair’s binding sites, which are at positions −26 bp and +26 bp relative to the center of the two re-TALEN-binding sites. For Cas9-gRNA and ssODN graphs, the green dashed lines mark the outer boundary of the gRNA targeting site, which are at positions −20 and −1 bp relative to the Protospacer Associated Motif sequence. Bottom: Deletion/Insertion size distribution in hiPSCs analyzed from the entire NHEJ population with treatments indicated earlier in the text. (c) The genome-editing efficiency of re-TALENs and Cas9-gRNAs targeting CCR5 in PGP1 hiPSCs. Top: schematic representation of the targeted genome-editing sites in CCR5. The 15 targeting sites are illustrated by blue arrows later in the text. For each site, cells were co-transfected with a pair of re-TALENs and their corresponding ssODN donor carrying 2 bp mismatches against the genomic DNA. Genome-editing efficiencies were assayed 6 days after transfection. Similarly, we transfected 15 Cas9-gRNAs with their corresponding ssODNs individually into PGP1-hiPSCs to target the same 15 sites and analyzed the efficiency 6 days after transfection. Bottom: the genome-editing efficiency of re-TALENs and Cas9-gRNAs targeting CCR5 in PGP1 hiPSCs. Panels 1 and 2 indicate NHEJ and HDR efficiencies mediated by reTALENs. Panels 3 and 4 indicate NHEJ and HDR efficiencies mediated by Cas9-gRNAs. NHEJ rates were calculated by the frequency of genomic alleles carrying deletions or insertions at the targeting region; HDR rates were calculated by the frequency of genomic alleles carrying 2 bp mismatches. Panel 5, the DNaseI HS profile of a hiPSC cell line from ENCODE database (Duke DNase HS, iPS NIHi7 DS). Of note, the scales of different panels are different.

Figure 3.

Study of functional parameters governing ssODN-mediated HDR with re-TALENs or Cas9-gRNAs in PGP1 hiPSCs. (a) PGP1 hiPSCs were co-transfected with re-TALENs pair (#3) and ssODNs of different lengths (50, 70, 90, 110, 130, 150 and 170 nt). All ssODNs possessed an identical 2 bp mismatch against the genomic DNA in the middle of their sequence. A 90 mer ssODN achieved optimal HDR in the targeted genome. The assessment of HDR, NHEJ-incurred deletion and insertion efficiency is described in the ‘Materials and Methods’ section. (b) 90 mer ssODNs corresponding to re-TALEN pair #3 each containing a 2 bp mismatch (A) in the center and an additional 2 bp mismatch (B) at different positions offset from A (where offsets varied from −30 to 30 bp) were used to test the effects of deviations from homology along the ssODN. Genome-editing efficiency of each ssODN was assessed in PGP1 hiPSCs. The bottom bar graph shows the incorporation frequency of A only, B only and A + B in the targeted genome. HDR rates decrease as the distance of homology deviations from the center increase (see text and Supplementary Figure S7a and b). (c) ssODNs targeted to sites with varying distances (−620∼480 bp) away from the target site of re-TALEN pair #3 were tested to assess the maximum distance within which we can place ssODNs to introduce mutations. All ssODNs carried a 2 bp mismatch in the middle of their sequences. We observed minimal HDR efficiency (≤0.06%) when the ssODN mismatch was positioned 40 bp away from the middle of re-TALEN pair’s binding site. (d) PGP1 hiPSCs were co-transfected with Cas9-gRNA (AAVS1) and ssODNs of different orientation (O_c: complement to gRNA; O_n: non-complement to gRNA) and different lengths (30, 50, 70, 90 and 110 nt). All ssODNs possessed an identical 2 bp mismatch against the genomic DNA in the middle of their sequence. A 70 mer O_c achieved optimal HDR in the targeted genome.

Figure 4.

Using re-TALENs and ssODNs to obtain monoclonal genome-edited hiPSC without selection. (a) Timeline of the experiment. (b) Genome engineering efficiency of re-TALENs pair and ssODN (#3) assessed by the NGS platform described in Figure 2b. (c) Sanger sequencing results of monoclonal hiPSC colonies after genome editing. Of note, the 2 bp heterogeneous genotype (CT/CT→TA/CT) was successfully introduced into the genome of PGP1-iPS-3-11, PGP1-iPS-3-13 colonies. (d) Immunofluorescence staining of targeted PGP1-iPS-3-11. Cells were stained for the pluripotency markers Tra-1-60 and SSEA4. (e) Hematoxylin and eosin staining of teratoma sections generated from monoclonal PGP1-iPS-3-11 cells.