In vitro transcribed sgRNA causes cell death by inducing interferon release

The clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated proteins 9 (Cas9) systems are powerful tools for gene editing. Ribonucleoprotein (RNP) complex composed of Cas9 protein and sgRNA binds to specific genomic loci and generate DNA double strand breaks. While plasmids expressing Cas9 protein and sgRNA are routinely transfected into various cell lines to perform gene editing (Cong et al., 2013; Mali et al., 2013; Ran et al., 2013), direct delivery of Cas9-sgRNA RNP has shown higher efficiency and lower off-target effects (Kim et al., 2014), especially in human primary cells such as T cells (Hendel et al., 2015; Schumann et al., 2015). SgRNA can be generated by either in vitro transcription (IVT) or chemical synthesis. IVT is widely used to generate sgRNAs, since it can be easily performed in most labs. Gene editing of human primary cells, such as human CD34 hematopoietic stem and progenitor cells (HSPCs), T cells and chimeric antigen receptor T (CAR-T) cells, is important for studying gene functions in these cell types, and holds great promise to further improve cell therapy (Schumann et al., 2015; Dever et al., 2016; Liu et al., 2016; Ren et al., 2017; Zhang et al., 2017). Our previous study showed that multiplex gene editing using CRISPR-Cas9 RNP hampered the proliferation of CAR-Tcells (Liu et al., 2016). It was also reported that Cas9/hCD45sg1 RNP-treated human HSPCs had lower cell number compared to Cas9 proteintreated control (Gundry et al., 2016). In this study, we investigated the mechanism of cell loss after electroporation of Cas9-sgRNA RNP and find an easy method to resolve it. As our initial attempt of gene editing in human CD34 HSPCs, we delivered Cas9-sgRNA RNPs into HSPCs by electroporation, using sgRNA in vitro transcribed by T7 polymerase. We observed significantly decreased cell number and reduced CD34 expression in survived cells 48 h after electroporation in experiments using five different sgRNA (Fig. S1A and S1B). Accordingly, the colony forming ability of HSPCs after RNP electroporation was markedly compromised (Fig. S1C). To identify the factor that contributed to this effect, we delivered IVT sgRNA (sgRNA-IVT), RNP complex consisting of Cas9 protein and IVT sgRNA (RNP-IVT), or Cas9 protein alone individually into primary HSPCs by electroporation. We observed lower cell viability, reduced CD34 expression and decreased colony forming ability in sgRNA-IVT and RNP-IVT groups, while the cells in Cas9 protein group and mock electroporation group survived well (Fig. 1A–C), indicating that sgRNA-IVT reduced HSPC stemness in addition to causing cell death. To validate whether sgRNA could also cause the death of T cells, we electroporated different amounts of IVT sgRNA into human primary CD3 T cells. Reduced cell viability and increased immune stimulation were observed when higher amounts of sgRNAs were used (Fig. S2A). All sgRNAs we tested had a negative effect on the survival of HSPCs and T cells, suggesting that some common feature of these IVT sgRNA was responsible for this effect. Based on existing reports, we hypothesized that the 5’ triphosphate of IVT sgRNA induced type I IFN production, leading to cell death in HSPCs and T cells. To test this hypothesis, we electroporated sgRNA-IVT targeting different genomic loci into CD34 HSPCs and CD3 T cells respectively, and measured the concentration of type I IFN in the culture medium using enzyme-linked immunosorbent assay (ELISA). Indeed, we detected significant release of IFN I in all samples electroporated with RNP-IVT (Figs. 1D and S2B). Next we explored the effects of type I IFN on human primary cells, we cultured freshly isolated HSPCs or CD3 T cells with IFN I at different concentration. We found that the survival and CD34 expression of HSPC were decreased drastically along with the increased concentration of IFN-α. However, IFN-β had no obvious effect (Fig. 1E and 1F). Similar results were obtained in CD3 T cells (Fig. S3A). Using Caspase-Glo 3/7 assay, we detected significantly increased caspase activity in T cells cultured with IFN-α (Fig. S3B). These data confirmed that IVT sgRNA induced IFN-α release, which led to the apoptosis of primary cells. To verify whether the 5’ triphosphate of sgRNA induced the production of IFN I, we removed the 5’ triphosphates of sgRNA using calf intestine phosphatase (CIP) (Dong-Ho et al., 2004) and chemically synthesized a 5’-hydroxylated (5’-OH) sgRNA targeting AAVS1 site. The viability of HSPCs and T cells electroporated with CIP-treated and chemically synthesized sgRNA together with Cas9 protein were similar to control, which was significantly higher than RNP-IVT


Cell Lines. Human 293T and Hela cells were maintained in DMEM (Gibco)
supplemented with 10% (v/v) FBS, 100 U/mL penicillin and streptomycin. The CD19 and luciferase expressing K562 and Jukat cells were maintained in RPMI1640 medium (Gibco) supplemented with 10% (v/v) FBS, 100 U/mL penicillin and streptomycin. All cell lines were cultured at 37 °C in a 5% CO 2 atmosphere.

Isolation of CD3 + T cells and CD34 + HSPCs from umbilical cord blood (UCB)
units. Fresh UCB units were obtained from healthy volunteer donors who have provided informed consent from the Beijing Cord Blood Bank (Beijing, China), and mononuclear cells were separated with human mononuclear cells separation medium 1.007 (Beijing DongFangHuaHui Biomedical technology co., Ltd). T cells were isolated using the EasySep human T cell enrichment kit (Stemcell Technologies), activated and expanded with anti-CD3/anti-CD28 Dynabeads (Thermo Fisher Scientific) at the ratio of 1:1 according to the manufacturer's instructions. T cells were cultured in X-vivo15 medium (Lonza) supplemented with 5% (v/v) heat-inactivated fetal bovine serum (Gibco) and 300 IU/mL recombinant human IL-2 (Sino Biological Inc.). HSPCs were isolated using the human cord blood CD34 positive selection kit II (Stemcell Technologies), and cultured in stem span H3000 with expansion supplement (Stemcell Technologies). All cells were cultured at 37 °C in a 5% CO 2 atmosphere.

Generation of CAR-T cells.
Anti-CD19 CAR-T cells were generated and expanded as previously described (Liu et al., 2016) with minor modification. Briefly, freshly purified primary CD3 + T cells were activated for 24 h and then infected with lentiviruses harboring the anti-CD19 CAR. Lentiviruses were produced by co-transfecting lentiviral vector with packaging plasmids pMD2.G, psPAX2 into 293T cell and the virus supernatant was harvested 48h post transfection.

In vitro transcription.
The plasmid PX330 (#42230) containing sgRNA backbone was used as PCR template and the acquired PCR amplicon containing T7 promoter, 20bp target sequence and sgRNA backbone was used as IVT template. The in vitro transcription was performed using MEGAshortscript T7 kit (Thermo Fisher Scientific). For the CIP (NEB) treatment, 2U enzyme was added to each μg of in vitro transcribed sgRNA, and further incubated at 37 °C for one hour. SgRNA were then purified with MEGAclear columns (Thermo Fisher Scientific) and eluted with elution buffer.
Electroporation of Human Primary Cells. Cas9 and sgRNA ribonucleoprotein (RNP) were prepared immediately before electroporation by incubating 6 μg Cas9 protein (provided by Shenzhen Fapon Biological Therapy Co., Ltd) with 6 μg indicated sgRNA at room temperature for 20 min. 1×10 5 cells were centrifuged at 200 g for 5 minutes and resuspended in 20 µl transfection buffer containing indicated RNP or sgRNA alone and then transferred into the electroporation cuvette. All electroporation experiments were performed using  and the P3 Primary Cell 4D-Nucleofector X Kit (V4XP-3024, Lonza), program EO-115 and program EO-100 was selected for CD3 + T or CD34 + HSPCs respectively.
After electroporation, cells were resuspended in 200 µl pre-warmed medium and transferred into a 96-well cell plate and incubated at 37 °C in an atmosphere of 5% CO 2 .  HSPCs or AAVS1 in 293T, Hela and Jukat T cells were determined by surveyor nuclease assay using surveyor mutation detection kit (Integrated DNA Technologies, Inc). The percentage of target disruption was quantified by densitometry and calculated as described (Guschin et al., 2010). The indels frequency of TRAC and B2M were measured by TIDE (Tracking Indels by Decomposition) analysis (Brinkman et al., 2014) in CAR-T cells. The PCR primers used for the amplification of target loci are listed in Supplementary Table 1.
Cytokine enzyme-linked immunosorbent assay (ELISA). The amount of interferon α and β secreted into the growth medium was determined using IFN-α (Biolegend) and IFN-β (PBL) ELISA kits. The medium from indicated cells was collected 24 h after the electroporation and was assayed according to the manufacturer's protocols.
Cytokines (IFN-γ，IL-2) production by effector (CAR T, KO CAR T, T) cells were evaluated by ELISA (Biolegend) according to the manufacturer's protocols.
Luciferase-based cytolysis assay. K562-CD19-luciferase cell based cytotoxicity was performed by a modified version of a luciferase-based CTL assay (Moon et al., 2014).
Briefly, K562-CD19-luciferase cells and effector cells were suspended at a density of 4×10 5 cells/ml in RPMI1640 medium, then seeded in white opaque plate at the ratio of 1:1 and incubated at 37°C in 5% CO 2 for 16 h. 10 µl of Steady-Glo luciferase substrate (Promega) was added, 5 min later, luminescence was recorded by PerkinElmer VICTOR X3. The results were reported as percentage of killing based on the luciferase activity in the wells compared with tumor cells alone (% killing = 100 -((RLU from well with effector and target cell coculture) / (RLU from well with target cells) × 100)).
Colony Forming Unit Assay. 1000 viable HSPCs were suspended immediately or 48 h post electroporation in 300 μl IMEM (Gibco) supplemented with 2% FBS, and then added the cell mix into 3 ml H4434 Methoculture TM medium (Stemcell Technology).
Cells were mixed and seeded into two wells of a 6-well Smartdish (Stemcell Technology), which were then cultured at 37 °C with saturated humidity and an atmosphere of 5% CO 2 . Colonies data were collected and analyzed by STEMvision (Stemcell Technology) two weeks later.
In vivo chimeric assay. 5-6 weeks old NOD-Prkdcscid Il2rgnull (NPG) mice (VITALSTAR, Beijing, China) were injected with 1×10 5 electroporated HPSCs via intravenous injection after 1.6 Gy X-ray irradiation. Peripheral blood and bone marrow were collected 12 weeks and 16 weeks after transplantation. Mononuclear cells were stained with anti-mouse CD45 APC and anti-human CD45-PE antibodies (Biolegend) after red blood cell lysis. The chimeric efficiency were calculated with the following formula: chimeric % = human CD45 positive %/ (human CD45 positive % + mouse CD45 positive % ). Caspase-Glo R 3/7 Assay. Caspase 3/7 activity was determined using Caspase-Glo R 3/7 assay (Promega) with modified protocol. Briefly, we used 10,000 cells per well in a 96-well plate with 100 μl medium. 12 hours after co-culture of T cells with IFN-α (100pg/ml) or 24 hours post electroporation of HSPCs and T cells,96-well plates containing cells were removed from the incubator and equilibrated to room temperature.100 μl Caspase-Glo R 3/7 reagent was added into each well. The plates were incubated at room temperature for 30 minutes after gentle mix. The luminescence of each sample was recorded by PerkinElmer VICTOR X3.