CRISPR-Cas12a-integrated transgenes in genomic safe harbors retain high expression in human hematopoietic iPSC-derived lineages and primary cells

Abstract

Discovery of genomic safe harbor sites (SHSs) is fundamental for multiple transgene integrations, such as reporter genes, chimeric antigen receptors (CARs), and safety switches, which are required for safe cell products for regenerative cell therapies and immunotherapies. Here we identified and characterized potential SHS in human cells. Using the CRISPR-MAD7 system, we integrated transgenes at these sites in induced pluripotent stem cells (iPSCs), primary T and natural killer (NK) cells, and Jurkat cell line, and demonstrated efficient and stable expression at these loci. Subsequently, we validated the differentiation potential of engineered iPSC toward CD34⁺ hematopoietic stem and progenitor cells (HSPCs), lymphoid progenitor cells (LPCs), and NK cells and showed that transgene expression was perpetuated in these lineages. Finally, we demonstrated that engineered iPSC-derived NK cells retained expression of a non-virally integrated anti-CD19 CAR, suggesting that several of the investigated SHSs can be used to engineer cells for adoptive immunotherapies.

Keywords: Cell biology; Molecular Genetics; Molecular biology; Stem cells research; Techniques in genetics.

Graphical abstract

Figure 1

Optimization of electroporation conditions, selection of SHS, and validation (A) Screen for optimal EP conditions for KOLF2-C1 iPSCs: 31 programs × 5 buffers (155 EP conditions), using MAD7^3×NLS and crDNMT1 (biological replicas n = 1; technical replicas t = 1). Indel frequency (analyzed by targeted NGS) as a function of EP programs. (B) Indel frequency of SHS crRNAs (from C) as a function of PAM sequence. Boxplot generated by ggplot2. Error bars represent standard deviation (SD) of indel frequency among crRNAs that have the same PAM sequence. Dots beyond the end of the whiskers represent outliers. (C) Indel frequency as a function of SHS crRNAs. crRNA specificity score is depicted as dots. KOLF2 were electroporated in buffer P3, using program CA137, with MAD7^3×NLS and SHS crRNA RNPs (n = 1; t = 1). See also Figure S1E for a biological replica of SHS crRNA screen using MAD7^1×NLS. (D) Validation of indel frequency as a function of top crRNAs (n = 3–4; t = 1–2). Error bars represent SD among biological replicas. (E) Validation of crAAVS1_3 indel frequency in iPSCs and Jurkat (n = 1; t = 3). Error bars represent SD among technical replicas. (F) Representative CRISPResso2-derived nucleotide percentage quilt around 4 selected SHS crRNAs and histograms depicting the size and frequency of insertions, deletions, and substitutions. Not the entire sequence of crRNA is shown in the quilt because the quantification window is set to ±10 bases around the MAD7 cut site (+1 base at the 3′ end of crRNA, as for Cas12a). (G) Relative mRNA expression levels of non-differentiation markers OCT4, SOX2, NANOG, and TDGF1 in samples transfected with the indicated crRNAs were calculated by the 2^−ΔΔCt method compared to GAPDH housekeeping gene and are shown here relative to a non-transfected control sample. Mean ± SD from qPCR technical quadruplicates is shown (n = 1, t = 4). See also Figure S1.

Figure 2

Transgene integrations into SHS in iPSCs, primary T and NK cells, and Jurkat (A) Quantification of GFP⁺ live iPS cells upon insertion of GFP into SHSs and ROSA26, 7 days after electroporation (post-EP). EP conditions: 2×10⁵ iPSC, buffer P3, program CA137, 100 pmol RNP (100:125 pmol MAD7^4×NLS:crRNA), 1 μg (∼0.45 pmol) of CAG-EGFP linear dsDNA HDRT. Non-targeting crRNAs crIDTneg1 and crIDTneg2 were used to assess the non-HDR-mediated insertion and expression of HDRT. (B) GFP median fluorescence intensity (MFI) in GFP⁺ cells from (A). (C) Comparison of integration efficiency using 0.5 μg (∼0.23 pmol) vs. 1 μg (∼0.45 pmol) of CAG-EGFP linear dsDNA HDRT in iPSCs, 9 days post-EP. (D) GFP MFI in GFP⁺ cells from (C). (E) Quantification of CAR⁺GFP⁺ double-positive, CAR⁺GFP^– single-positive, and total GFP⁺ iPSCs 5 days post-EP with 0.5 pmol CAG-CAR-EGFP HDRT or AAVS1_3 CAG-CAR HDRT. EP conditions: P3-CA137; 2×10⁵ iPSC, 50 pmol RNP (50:60 pmol MAD74xNLS:crRNA), 100 μg PGA (poly-L-glutamic acid) as RNP carrier, and 0.5 pmol corresponding linear dsDNA HDRT per reaction. (F) GFP MFI and CAR^myc MFI in GFP⁺/CAR⁺GFP⁺ iPSCs that are shown in (E). (G) Expression of CAG-CAR-EGFP (as CAR⁺GFP⁺ double-positive Jurkat cells) inserted into SHSs and ROSA26 in Jurkat over time (2, 8, 16 days post-EP). EP conditions: buffer SF, program CA137; 2×10⁵ Jurkat cells, 50 pmol RNP (50:75 pmol MAD7^4×NLS:crRNA, mixed with 100 μg PGA) and 0.5 pmol CAG-CAR-EGFP linear dsDNA HDRT per reaction. Control is IDTneg2 crRNA. Data are represented as mean ± SD of 3 technical replicas per sample from 1 biological replica (n = 1; t = 3); 2^nd biol. replica shown in Figure S2D. (H) Transfection of CAR-GFP in primary T cells and quantification of GFP⁺CAR⁺ live primary T cells on days 7 and 14 post-EP. T cells from two donors: T1 and T2, were electroporated in parallel; mean and SD from 3 technical replicas per crRNA-HDRT combination is shown (n = 2; t = 3). Control crRNA is IDTneg1. EP conditions: P3-EH115. More details in STAR Methods. (I) Expression of integrated CAR-GFP at SHS and ROSA26, and of CAR at AAVS1 in primary NK cells isolated from two donors NK3 and NK4 (n = 2; t = 3), at days 7 and 14 post-EP. Mean and SD from 3 technical replicas per crRNA-HDRT combination per donor are shown. Control is IDTneg1 crRNA. EP conditions: P3-EN138. More details in STAR Methods. See also Figure S2.

Figure 3

Hematopoietic differentiation of monoclonal iPSC lines with GFP/CAR-EGFP transgenes at SHSs (A) Differentiation overview (2D protocol; details in STAR Methods). (B) HSPCs derived from KOLF2. Initial seeding at different densities resulted in 4, 6 or 9 colonies/cm². HSPCs from each initial seeding category were pooled from 3 to 4 technical replica wells (t = 4, pooled), stained with viability marker/or antibodies recognizing the indicated key cell-surface markers of hematopoietic differentiation (CD34, CD43, CD45) in pairs, and analyzed by flow cytometry. (C) Quantification of positive cells from panel (B) upon double staining with CD34 and CD43 antibodies or CD43 and CD45 antibodies. (D) Monoclonal iPSC lines ROSA26_3 CAR-GFP (cl. A4), CXs313_4 GFP (cl. A8), or C7s301-5 GFP (cl. D9), along with parental KOLF2 were differentiated to HSPCs and harvested on day 11. Cells were stained with CD34, CD43, and CD45 antibodies, separately and pairwise. Staining controls: primary T cells, NALM6, iPSCs, and maturing HSPCs that were derived from iPSCs in a previous experiment. (E) Quantification of marker expression and GFP MFI in single-antibody-stained samples from D. “a” and “b” in sample name denote two technical replicas (t = 2). (F) The monoclonal iPSC lines C8s325_6 GFP (cl. H6) or C7s257_8 CAR-GFP (cl. B2 & D4) were differentiated to HSPCs and harvested on day 12. Cells were stained with CD34, CD43, and CD45 antibodies, separately and pairwise. Staining controls: primary T cells, NALM6, and maturing iPSC-derived HSPCs. (G) Quantification of marker expression and GFP MFI in single-antibody-stained samples from F. “a” and “b” in sample name denote two technical replicas (t = 2). See also Figures S3–S5.

Figure 4

Differentiation of monoclonal iPSC lines to lymphoid progenitor cells (LPCs) and NK cells (A and B) Parental and edited iPSC clones were differentiated to LPCs through initial CD34⁺ HSPC stage. HSPCs were reseeded and LPCs harvested on day 14 and stained with the pairwise antibody combinations indicated in Figure S6A. Quantification of cell subtypes from pairwise antibody staining. Data are from biological replica (n) = 1 and technical replica (t) = 1 per sample. (C and D) Parental KOLF2 iPSCs were differentiated to NK cells through the HSPC and LPC stages. The indicated samples obtained relatively sufficient NK number for analysis. Data are related to Figure S6C (n = 1; t = 1). (E and F) Parental KOLF2 iPSCs and iPSC lines CXs313_4 GFP (cl. A8) and ROSA26_3 CAR-GFP (cl. A4) were differentiated to NK cells which were analyzed by immunostaining and flow cytometry (n = 1; t = 1). (G) Parental KOLF2 iPSCs and the iPSC clones C7s257 CAR-GFP (cl. B2 and cl. D4) and C8s325 GFP (cl. H6) were initially differentiated to CD34⁺ HSPCs (see Figures 3F and 3G), followed by differentiation to NK cells (abbreviated here iNK) (n = 1; t = 1). Cells were harvested at 29 days and stained with antibodies in pairwise combinations. CD56⁺CD45^+bright are considered as NK cells. (H and I) Quantification of cells positive for key markers (n = 1; t = 1). Samples are from same experiment as in panel (G). (J) Cell apoptosis assay using 10⁴ NALM6-RFP⁺ (expressing red fluorescent protein; RFP) as target cells. The apoptosis reagent IncuCyte Annexin V Green was added (final dilution 1:200) into the target cells 1 h after seeding, followed by immediate addition of the indicated effector cells in different ratios (K = 10³ cells). Effector cells were primary NK cells from donor NK2 (15 days after isolation from blood and kept in culture), KOLF2-derived NK cells and NK derived from iPSC line ROSA26_3 CAR-GFP (cl. A4). As positive control, absolute ethanol was added to 3 replicate wells to a final concentration 10%, 2 h after initial target cell seeding. NK Basal Medium was used for all cell suspensions in the assay. Each treatment consists of 3 technical replica wells. The graph shows the average ratio of NALM6 RFP⁺AnnexinV⁺/NALM6 RFP⁺ and SD of 3 technical replicas (t = 3). The assay was done as one biological replica (n = 1). Cells were imaged by IncuCyte system at 20× magnification by non-adherent cell-by-cell format. See also Figure S6.