MAGIK: A rapid and efficient method to create lineage-specific reporters in human pluripotent stem cells

Introduction: Precise insertion of fluorescent proteins into lineage-specific genes in human pluripotent stem cells (hPSCs) presents challenges due to low knockin efficiency and difficulties in isolating targeted cells. To overcome these hurdles, we present the modified mRNA (ModRNA)-based Activation for Gene Insertion and Knockin (MAGIK) method. MAGIK operates in two steps: first, it uses a Cas9-2A-p53DD modRNA with a mini-donor plasmid (without a drug selection cassette) to significantly enhance efficiency. Second, a deactivated Cas9 activator modRNA and a 'dead' guide RNA are used to temporarily activate the targeted gene, allowing for live cell sorting of targeted cells. Consequently, MAGIK eliminates the need for drug selection cassettes or labor-intensive single-cell colony screening, expediting precise gene editing. We showed MAGIK can be utilized to insert fluorescent proteins into various genes, including SOX17, NKX6.1, NKX2.5, and PDX1, across multiple hPSC lines. This underscores its robust performance and offers a promising solution for achieving knockin in hPSCs within a significantly shortened time frame.

Materials and

Methods: ModRNA or plasmid-based knock-in in hPSCs
For knock-in, ~14,000 cells/cm2 hPSCs were seeded onto iMatrix-511 coated wells of a 12-well plate and cultured for 24 hours at 37°C, 5% CO2. For modRNA-based knock-in, iCas9 modRNA, KI sgRNA, and mini-donor plasmid were combined with Lipofectamine Stem Transfection Reagent (ThermoFisher) (1:4 ratio, mass/volume) in Opti-MEM media. For plasmid-based knock-in, iCas9 modRNA was replaced with the EFS-Cas9 plasmid. Before transfection, the spent culture medium was replaced with fresh TeSR supplemented with 10 μM Y-27632. The transfection mix was incubated at room temperature for 10 minutes before being added to the well. The transfection media was changed 24 hours later, and cells were maintained in TeSR with daily media changes.

ModSAM-mediated target gene activation in hPSCs
For modSAM-mediated gene activation, ~28,000 cells/cm2 hPSCs were seeded onto iMatrix-511 coated wells of a 12-well plate and cultured for 24 hours at 37°C, 5% CO2. For modRNA-based activation, the dCas9 activator modRNA and dgRNA were combined with Lipofectamine Stem Transfection Reagent (1:4 ratio, mass/volume) in Opti-MEM medium. For plasmid-based activation, the dCas9 activator modRNA was replaced with pPB-R1R2-EF1a-VP64dCas9VP64-T2A-MS2p65HSF1-IRES-bsd-pA plasmid (Addgene, #113341). Before transfection, the spent culture medium was replaced with fresh TeSR supplemented with 10 μM Y-27632. The transfection mix was incubated at room temperature for 10 minutes before being added to the well in a dropwise fashion followed by a media change 24 hours later. Cells were collected between 36 and 48 hours after transfection for either fluorescence activated cell-sorting (FACS) or flow cytometry.

Results, Conclusions, and Discussions: Here we developed a modRNA-based synergistic activation mediator (modSAM) system (Fig. 1A). By applying our modSAM system, we managed to achieve robust activation of lineage-specific genes, NKX6.1, PDX1, and NKX2.5 in hPSCs (Fig. 1B-D). Next, we decided to evaluate whether hPSCs maintain pluripotency 5 days post modSAM transfection by analyzing the expression of pluripotency markers. Nearly 100% of day 5 cells were positive for OCT4, NANOG and SOX2 based on immunostaining assays (Fig 1E). This was confirmed by flow cytometry analysis (Fig 1F-G).

Considering the transient nature of the activation and the absence of differentiation in the modSAM transfected cells, we hypothesized that our modSAM system might serve as a selection tool for purifying targeted cells after Cas9-mediated knockin of fluorescent proteins to lineage-specific genes.

Next, we decided to use our MAGIK method to create an NKX6.1-nEGFP H9 reporter line (Fig 2A-B). H9 cells were initially transfected with iCas9 modRNA, NKX6.1 KI sgRNA, and NKX6.1-nEGFP mini-donor plasmid. After a cultivation period, cells were passaged and transfected with our modSAM system (dCas9 activator modRNA + NKX6.1-dgRNA4). Two days post-transfection, EGFP+ cells were sorted and replaced. The purity of EGFP+ cells was 3.63% (Fig. 2C). To study whether the sorted cells retained pluripotency, we assessed the cells via immunostaining of pluripotency makers. Our data showed the sorted cells exhibited nearly 100% expression of OCT4, NANOG, and SOX2 (Fig 2D), with flow cytometry revealing similar expression levels of OCT4 and TRA-1-60 to WT H9 cells (Fig 2E). From the sorted cell population, we derived single cell clones and extracted genomic DNA (gDNA). PCR on the gDNA with our 5’ Out and 3’ Out primers confirmed successful integration of EGFP at the expected site in all clonal populations (Fig. 2F). We further demonstrated that we can use MAGIK to do knockin for SOX17, PDX1 and NKX2.5 in multiple hPSC lines.

In summary, the MAGIK method represents a significant step forward in the field of stem cell research. Its efficiency, simplicity, and applicability across various genes and stem cell lines make it a promising tool for developing lineage-specific reporters.

Acknowledgements (Optional): This work was supported by NSF CBET-1943696 (to X.L.L.), NSF CBET-2143064 (to X.B.), NIH R21EB026035 (to X.L.L.), NIH R56DK133147 (to X.L.L.), Huck Innovative and Transformational Seed (HITS) Fund (to X.L.L.), and NIH R37CA265926 (to X.B.).