In vitro transcribed sgRNA causes cell death by inducing interferon release
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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-T cells (Liu et al., 2016). It was also reported that Cas9/hCD45sg1 RNP-treated human HSPCs had lower cell number compared to Cas9 protein-treated 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.
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-GloR 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 samples (Fig. S4A and S4B). Consistently, the caspase activity induced by sgRNA without 5’ triphosphate was much lower compared with sgRNA containing 5’ triphosphate (Fig. S4C and S4D). These results demonstrated that the 5’ triphosphate of sgRNA is the major factor that induced innate immunity and primary cell death.
Similar to HSPCs, CIP treatment of IVT RNP (RNP-CIP) targeting various genomic loci also attenuated immune-stimulation and improved cell viability of CD3+ T cells and anti-CD19 CAR-T cells (Figs. S7 and S8). These results demonstrated that the 5’ triphosphate of IVT sgRNA induced type I IFN production, which led to cell death; removing the 5’ triphosphate using CIP could rescue these detrimental effects.
We also explored the underlying mechanism of IVT sgRNA induced IFN I release using HeLa cells. We observed the phosphorylation of IRF3 and the expression levels of IFNB1, IFIT1, MDA5 and RIG-I were significantly up-regulated in sgRNA-IVT and RNP-IVT electroporation groups (Figs. S9A, S9B, S9C and S10), confirming the innate immunity was also activated in HeLa cells.
Next, we established MDA5 and RIG-I knockout HeLa cell lines (Fig. S9D). Compared with wild type (WT), the expression of IFNB1 and IFIT1 in MDA5 KO was markedly decreased, and was comparable to mock, RIG-I KO and MDA5/RIG-I DKO clones after IVT sgRNA electroporation (Fig. S9E). These results suggested that MDA5 and RIG-I recognized the IVT sgRNA and induced IFN expression.
We next assessed the effect of removing 5’ triphosphate of IVT sgRNA via CIP on gene editing efficiency. Firstly, we found that the removal of 5’ triphosphate did not significantly affect the persistence of sgRNA in 293T cells (Fig. S11A). We further analyzed the gene editing efficiency of sgRNA-CIP, and found that CIP treatment did not affect the gene editing efficiency of the CRISPR-Cas9 system in HSPCs, T cells, CAR-T cells and various cell lines (Figs. S11B, S11C and S12). Furthermore, performing multiplex gene editing in anti-CD19 CAR-T cells using sgRNA -CIP did not affect the function of CAR-T cells (Fig. S13).
In this study, we showed that the triphosphate at 5’ end of IVT sgRNA triggered the innate immunity through RIG-I/MDA5 signaling pathway and induced the production of type I IFN, which led to the death of CD34+ HSPCs and CD3+ T cells, and stemness reduction in CD34+ HSPCs. By treating the IVT sgRNA with CIP, we were able to eliminate the IFN induction effect of IVT sgRNA, and achieved significantly better cell survival without affecting gene editing efficiency of the CRISPR-Cas9 system. While our manuscript is under review, another group published similar findings in HeLa and T cells, and identified RIG-I as the sensor for IVT sgRNA (Kim et al., 2018). In addition to T and CAR-T cells, our work demonstrated a dramatic effect on both the survival and stemness of human HSPCs, and identified MDA5 as another player involved in innate immunity response induced by IVT sgRNAs.
Although sgRNA generated by chemical synthesis is an option, the generally high costs and long production time limit their widespread application. Moreover, the length of sgRNA is almost reaching the current limits of RNA chemical synthesis. With various optimization and added structures and sequences, sgRNAs longer than 100 nt are commonly used (Shechner et al., 2015; Cheng et al., 2016) and are most conveniently generated by IVT. Therefore, RNA-CIP is useful for a variety of applications in gene editing and transcription regulation of primary cells.
We would like to thank Junning Wei and Yi Yang (Beijing Cord Blood Bank) for their help in preparing the cord blood samples. We would also like to thank Lei Shi (Beijing Cord Blood Bank) for her help with CFU analysis. This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16010205), National Key Research and Development Program of China (2018YFA0107703), and the National Natural Science Foundation of China (Grant Nos. 81773269 and 31722036). HW is supported by the “Young Thousand Talent Project”.
H. Wang conceived and supervised the entire project. W. Mu performed experiments in T and CAR-T cells. N.T. and C.C. performed electroporation of HSPCs and prepared cells for colony formation assays. X. Wei provided human umbilical cord blood. W. Mu, N. Tang, W. Sun, and H. Wang analyzed the data and wrote the manuscript.
Wei Mu, Na Tang, Chen Cheng, Wen Sun, Xiaofei Wei and Haoyi Wang declare that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000 (5). All donors of umbilical cord blood (UCB) units have provided informed consent. All institutional and national guidelines for the care and use of laboratory animals were followed.
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