MSCV-based retroviral plasmids expressing&nbsp;3xFLAG-Sp-dCas9&nbsp;for enChIP analysis

doi:10.21203/rs.2.16753/v6

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MSCV-based retroviral plasmids expressing 3xFLAG-Sp-dCas9 for enChIP analysis

https://doi.org/10.21203/rs.2.16753/v6

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published 08 Jul, 2021

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Engineered DNA-binding molecule–mediated chromatin immunoprecipitation (enChIP) is a technology for purifying specific genomic regions to facilitate identification of their associated molecules, including proteins, RNAs, and other genomic regions. In enChIP, the target genomic region is tagged with engineered DNA-binding molecules, e.g., a variant of the clustered regularly interspaced short palindromic repeats (CRISPR) system consisting of a guide RNA (gRNA) and a catalytically inactive form of Cas9 (dCas9). In this study, to increase the flexibility of enChIP and expand the range of target cells, we generated murine stem cell virus (MSCV)-based retroviral plasmids for expressing dCas9. We constructed MSCV-based retroviral plasmids expressing Streptococcus pyogenes dCas9 fused to a 3xFLAG-tag (3xFLAG-Sp-dCas9) and various drug resistance genes. We showed that by using these plasmids, it is feasible to purify target genomic regions with yields comparable to those reported using other systems. These systems might give enChIP users greater flexibility in choosing optimal systems for drug selection of transduced cells.

Epigenetics & Genomics

enChIP

dCas9

ChIP

chromatin immunoprecipitation

CRISPR

MSCV

To understand the molecular mechanisms underlying regulation of genome functions such as epigenetic regulation and transcription, it is necessary to identify the regulatory molecules that bind to a genomic region of interest. We recently developed engineered DNA-binding molecule–mediated chromatin immunoprecipitation (enChIP) technology for the isolation of genomic regions of interest to facilitate identification of their associated molecules (1). The engineered DNA-binding molecules that can be used to tag a target locus include transcription activator-like (TAL) proteins (2), and the clustered regularly interspaced short palindromic repeats (CRISPR) system (3-5) consisting of a guide RNA (gRNA) and a catalytically inactive form of Cas9 (dCas9) (see our recent review (6) for a comprehensive list of publications using CRISPR-based systems). Locus-tagging can be achieved by expression of engineered DNA-binding molecules in the cells to be analyzed (in-cell enChIP) (1). Alternatively, it can be achieved in vitro using recombinant or synthetic engineered DNA-binding molecules (in vitro enChIP) (7). After tagging with an engineered DNA-binding molecule, the locus is isolated by affinity purification and its associated proteins are identified by mass spectrometry (MS) (1,8). Associated nucleic acids such as RNAs (9,10) and other genomic regions (11,12) are identified by next-generation sequencing (NGS).

In this study we generated murine stem cell virus (MSCV)-based retroviral plasmids expressing 3xFLAG-Sp-dCas9 and various drug resistance genes. Using these plasmids, we were able to purify target genomic regions with yields comparable to those reported using other systems. These systems might give enChIP users greater flexibility in choosing optimal systems for drug selection of transduced cells. In addition, they could be used to analyze different types of target cells.

Plasmids

To construct MSCV expression plasmids expressing 3xFLAG-Sp-dCas9, the pMSCVneo, pMSCVhyg, and pMSCVpuro vectors (631461, Takara Bio, Kusatsu, Japan) were digested with BglII (1021A, Takara Bio) and Xho I (1094A, Takara Bio). After a blunting reaction, the plasmids were treated with bacterial alkaline phosphatase (E. coli C75) (2120A, Takara Bio). The cleaved vectors were purified by agarose gel electrophoresis and ligated with the coding sequence of 3xFLAG-Sp-dCas9, which was isolated from 3xFLAG-dCas9/pMXs-puro (Addgene (Watertown, MA, USA) #51240) (13) by digestion with Pac I (R0547, New England Biolabs, Ipswich, MA, USA) and Not I (1166A, Takara Bio).

To construct gRNA-hIRF-1 #12/pSIR-hCD2 (Addgene #135392), pSIR-hCD2 (Addgene #51143) (13) was digested with EcoR I (1040A, Takara Bio) and treated with bacterial alkaline phosphatase. The cleaved vector was purified by agarose gel electrophoresis and ligated with the gBlock targeting the interferon (IFN) regulatory factor (IRF)-1 gene promoter, which was isolated from gRNA-hIRF-1 #12 (Addgene #61079) (1) by digestion with EcoR I.

A retroviral vector expressing a gRNA targeting the promoter region of the mouse c-myc gene, m-c-myc gRNA #1/pSIR-hCD2, was previously described (14).

The Addgene plasmid # of the newly made constructs are as follows: 3xFLAG-Sp-dCas9/pMSCVneo: 134982; 3xFLAG-Sp-dCas9/pMSCVhyg: 134323; 3xFLAG-Sp-dCas9/pMSCVpuro: 134983; gRNA-hIRF-1 #12/pSIR-hCD2: 135392.

Cell lines

The 293T cell line was derived by transformation of human embryonic kidney (HEK) 293 cells with the SV40 large T antigen (15). HT1080 is a human fibrosarcoma cell line purchased from ATCC (Manassas, VA, USA) (CCL-121) (16). 293T, HT1080, and HT1080-derived cells were cultured in DMEM (FUJIFILM Wako Pure Chemical, Osaka, Japan) supplemented with 10% fetal calf serum (FCS).

Transduction and transfection of retroviral plasmids

For transduction of retroviral plasmids expressing 3xFLAG-Sp-dCas9 into HT1080 cells, 5.5 µg of each plasmid was transfected into 1 × 10⁶ of 293T cells along with 5.5 µg of pPAM3 (17) using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA). Two days after transfection, viral supernatant was harvested and used for infection of HT1080 cells. Infected cells were selected in culture media containing G418 (Nacalai Tesque, Kyoto, Japan) (0.8 mg/ml), hygromycin (Nacalai Tesque) (75 µg/ml), or puromycin (Nacalai Tesque) (0.75 µg/ml).

For transduction of retroviral plasmids expressing the gRNA into HT1080-derived cells expressing 3xFLAG-dCas9, 5.5 µg of gRNA-hIRF-1 #12/pSIR-hCD2 was transfected into 1 × 10⁶ of 293T cells along with 5.5 µg of pPAM3 using Lipofectamine 3000. Two days after transfection, viral supernatant was harvested and used for infection of HT1080 cells expressing 3xFLAG-dCas9. Cells expressing 3xFLAG-Sp-dCas9 and gRNA were sorted by MACS as described below.

For transduction of retroviral plasmids expressing 3xFLAG-Sp-dCas9 into NIH 3T3 cells, 5.5 µg of each plasmid was transfected into 1 × 10⁶ of Plat-E cells (18) using Lipofectamine 3000. Two days after transfection, viral supernatant was harvested and used for infection of NIH 3T3 cells. Infected cells were selected in culture media containing G418 (0.7 mg/ml), hygromycin (100 µg/ml), or puromycin (1.5 µg/ml).

For expression of gRNA targeting the c-myc promoter into NIH 3T3-derived cells expressing 3xFLAG-dCas9, 4 x 10⁵ cells expressing 3xFLAG-dCas9 were plated in a well of 6-well plate. Next day, 4 µg of m-c-myc gRNA #1/pSIR-hCD2 was transfected using Lipofectamine 3000. Three days after transfection, cells were harvested for enChIP analysis.

Immunoblot analysis

Cytoplasmic extracts and nuclear extracts were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). Ten micrograms of cytoplasmic extract from HT1080-derived cells and eight micrograms of nuclear extracts from NIH 3T3-derived cells were subjected to immunoblot analysis with anti-FLAG M2 antibody (Ab) (F1804, Sigma-Aldrich, Saint Louis, MO, USA) as described previously (1).

MACS sorting and flow cytometry

MACS sorting of human CD2 (hCD2) (+) cells was performed using CD2 Microbeads (130-091-114, Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany). To monitor the results of MACS sorting, cells were stained with phycoerythrin (PE)-conjugated anti-hCD2 Ab (347597, BD Life Sciences, San Jose, CA, USA) for 30 min at 4°C. Flow cytometry was performed on a FACSCalibur (BD Life Sciences), and data were analyzed using the FlowJo software (BD Life Sciences).

enChIP-real-time PCR

enChIP-real-time PCR was performed as previously described (13). The primers used in this study were reported previously (1).

MSCV retroviral expression systems for enChIP

To increase the flexibility of enChIP and expand the range of target cells, we constructed retroviral plasmids expressing S. pyogenes dCas9 fused with the 3xFLAG-tag (3xFLAG-Sp-dCas9), using the MSCV system along with various drug selection markers (Fig. 1A). [insert Figure 1 here.]

To confirm the performance of the system, we transduced the retroviral plasmids into HT1080, a human fibrosarcoma cell line. After drug selection, expression of 3xFLAG-Sp-dCas9 was confirmed by immunoblot analysis with Ab against the FLAG-tag (Fig. 1B). Subsequently, gRNA-hIRF-1 #12/pSIR-hCD2, a self-inactivating retroviral vector expressing a sgRNA targeting human IRF-1 gene promoter and the hCD2 selection marker protein, was transduced into the HT1080-derived cells expressing 3xFLAG-Sp-dCas9. Two days after transduction, cells expressing the sgRNA (hCD2 (+) cells) were selected by MACS sorting. After expansion, 2 × 10⁶ cells were subjected to enChIP analysis. Briefly, the cells were crosslinked with formaldehyde, and the crosslinked chromatin was fragmented by sonication. Subsequently, fragmented chromatin tagged with the CRISPR complex was purified using anti-FLAG Ab. As shown in Fig. 2, the yields of enChIP were comparable to those reported using other systems (13). [insert Figure 2 here.]

We next examined if the MSCV retroviral expression system works in other cell lines and target loci. We transduced the retroviral plasmids into a mouse fibroblast cell line, NIH 3T3. After drug selection, expression of 3xFLAG-Sp-dCas9 was confirmed by immunoblot analysis with Ab against the FLAG-tag (Supplementary Fig. S1A). Subsequently, m-c-myc gRNA #1/pSIR-hCD2, an expression vector expressing a sgRNA targeting mouse c-myc gene promoter, was transfected into the NIH 3T3-derived cells expressing 3xFLAG-Sp-dCas9. Three days after transfection, cells were subjected to enChIP analysis. As shown in Supplementary Fig. S1B, the c-myc locus was specifically enriched by enChIP.

These results suggested that the enChIP system using the MSCV retroviral vectors can be used for purification of target genomic regions and subsequent downstream applications such as identification of molecules associated with the target genomic regions.

In this study, we developed enChIP systems using MSCV-based retroviral expression vectors with various selection markers. To the best of our knowledge, there is currently no retroviral plasmid expressing 3xFLAG-Sp-dCas9 and the hygromycin resistance gene in Addgene. These systems might give enChIP users greater flexibility in choosing optimal systems for drug selection of transduced cells. In addition, they could be used to analyze target cells that might be difficult to analyze using other systems.

Limitations

Although the tropism of MSCV-based retroviral vectors has been extensively analyzed, only one cell line (the HT1080 cell line) was analyzed in this study. In addition, although it has been shown that many different loci can be analyzed using 3xFLAG-Sp-dCas9, only one locus (the IRF-1 promoter) was targeted in this study. Further studies will be necessary to determine which cell types and which loci can be analyzed using this system. Further studies might also be necessary to assess the utility of these systems in combination with MS and NGS to identify molecules associated with target genomic regions.

In this study, we used hCD2 as a marker to select gRNA (+) cells. However, this might not work for cells expressing endogenous hCD2, such as ALL cell lines. Therefore, other selection markers should be used for these cells. In this regard, we have already developed and made available through Addgene other selection markers including drug resistance genes and fluorescent proteins such as green fluorescent protein and dsRed-Express2 red fluorescent protein (13). One of the advantages of hCD2 and other cell surface markers for selection is that cells expressing these markers can be purified directly using magnetic-based plasmid purification isolation systems and without the need for expensive instruments such as flowcytometric cell sorters.

enChIP: engineered DNA-binding molecule-mediated chromatin immunoprecipitation; CRISPR: clustered regularly interspaced short palindromic repeats; dCas9: a catalytically-inactive form of Cas9; gRNA: guide RNA; MS: mass spectrometry; NGS: next-generation sequencing; MSCV: murine stem cell virus; IFN: interferon; IRF-1: interferon regulatory factor-1; HEK: human embryonic kidney; FCS: fetal calf serum; Ab: antibody; hCD2: human CD2; PE: phycoerythrin; NLS: nuclear localization signal; CBB: Coomassie Brilliant Blue

Authors contributions

H.F. conceived the idea of development of enChIP systems using MSCV-based retroviral expression vectors with different selection markers and constructed expression plasmids. T.F. and H.F. designed and performed experiments and wrote the manuscript. M.Y. performed experiments. H.F. directed and supervised the study. All authors read and approved the final manuscript.

Funding

This work was supported by the Takeda Science Foundation (T.F.), a Grant-in-Aid for Scientific Research (C) (#15K06895, #18K06176) (T.F.), and a Grant-in-Aid for Scientific Research (B) (#15H04329) (T.F., H.F.), ‘Transcription Cycle’ (#15H01354) (H.F.), from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Conflict of interest statement

T.F. and H.F. are inventors of granted patents and a patent pending owned by Osaka University for technology relating to the purification and subsequent analysis of specific DNA, including genomic DNA with chromatin structure, using an engineered DNA-binding molecule including CRISPR complex binding to target DNA (Patent name: “Method for isolating specific genomic regions using molecule binding specifically to endogenous DNA sequence”, Patent numbers: Japan 5,954,808, EP 2,963,113; Patent application number: WO2014/125668). T.F. and H.F. are co-founders of Epigeneron, Inc. and own stock in the company. H.F. is one of directors of Epigeneron, Inc. H.F. is a member of the Advisory Board of Addgene.

Acknowledgements

We thank Dr. George M. Church for providing a plasmid (Addgene #43802).

1. Fujita, T. and Fujii, H. (2013) Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Biochem. Biophys. Res. Commun., 439, 132-136.

2. Bogdanove, A.J. and Voytas, D.F. (2011) TAL effectors: customizable proteins for DNA targeting. Science, 333, 1843-1846.

3. Harrison, M.M., Jenkins, B.V., O'Connor-Giles, K.M. and Wildonger, J. (2014) A CRISPR view of development. Genes Dev., 28, 1859-1872.

4. Wright, A.V., Nuñez, J.K. and Doudna, J.A. (2016) Biology and applications of CRISPR systems: harnessing nature's toolbox for genome engineering. Cell, 164, 29-44.

5. Xu, X. and Qi, L.S. (2018) A CRISPR-dCas Toolbox for Genetic Engineering and Synthetic Biology. J. Mol. Biol., 431, 34-47.

6. Fujita, T. and Fujii, H. (2019) Purification of specific DNA species using the CRISPR system. Biology Methods & Protocols, 4, bpz008.

7. Fujita, T., Yuno, M. and Fujii, H. (2016) Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins. Genes Cells, 21, 370-377.

8. Shamsi, F., Xue, R., Huang, T.L., Lundh, M., Liu, Y., Leiria, L.O., Lynes, M.D., Kempf, E., Wang, C.-H., Sugimoto, S. et al. (2020) FGF6 and FGF9 regulate UCP1 expression independent of brown adipogenesis. Nat. Commun., 11, 1421.

9. Fujita, T., Yuno, M., Okuzaki, D., Ohki, R. and Fujii, H. (2015) Identification of non-coding RNAs associated with telomeres using a combination of enChIP and RNA sequencing. PLoS One, 10, e0123387.

10. Zhang, Y., Hu, J.-F., Wang, H., Cui, J., Gao, S., Hoffman, A.R. and Li, W. (2017) CRISPR Cas9-guided chromatin immunoprecipitation identifies miR483 as an epigenetic modulator of IGF2 imprinting in tumors. Oncotarget, 8, 34177-34190.

11. Fujita, T., Yuno, M., Suzuki, Y., Sugano, S. and Fujii, H. (2017) Identification of physical interactions between genomic regions by enChIP-Seq. Genes Cells, 22, 506-520.

12. Seo, W., Shimizu, K., Kojo, S., Okeke, A., Kohwi-Shigematsu, T., Fujii, S.-i. and Taniuchi, I. (2020) Runx-mediated regulation of CCL5 via antagonizing two enhancers influences immune cell function and anti-tumor immunity. Nat. Commun., 11, 1562.

13. Fujita, T. and Fujii, H. (2014) Identification of proteins associated with an IFNγ-responsive promoter by a retroviral expression system for enChIP using CRISPR. PLoS One, 9, e103084.

14. Fujita, T., Kitaura, F., Oji, A., Tanigawa, N., Yuno, M., Ikawa, M., Taniuchi, I. and Fujii, H. (2018) Transgenic mouse lines expressing the 3xFLAG-dCas9 protein for enChIP analysis. Genes Cells, 23, 318-325.

15. DuBridge, R.B., Tang, P., HC, H., Leong, P.M., Miller, J.H. and Calos, M.P. (1987) Analysis of mutation in human cells by using an Epstein-Barr virus shttle system. Mol Cell Biol., 7, 379-387.

16. Rasheed, S., Nelson-Rees, W.A., Toth, E.M., Arnstein, P. and Gardner, M.B. (1974) Characterization of a newly derived human sarcoma cell line (HT-1080). Cancer, 33, 1027-1033.

17. Miller, A.D. and Buttimore, C. (1986) Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol. , 6, 2895-2902.

18. Morita, S., Kojima, T. and Kitamura, T. (2000) Plat-E: and efficient and stable system for transient packaging of retroviruses. Gene Ther., 7, 1063-1066.

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Journal Publication

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MSCV-based retroviral plasmids expressing 3xFLAG-Sp-dCas9 for enChIP analysis

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