Journal: bioRxiv
Article Title: Schlafen-11 and -9 are innate immune sensors for intracellular single-stranded DNA
doi: 10.1101/2024.02.24.581893
Figure Lengend Snippet: ( A to C ) Schematic overview of genome-wide CRISPR screening for essential genes in response to ODN60 (A). HEK293 cells were infected with a Brunello lentiviral pool containing Cas9 and a gRNA library. After selection with puromycin, infected cells were divided into an untreated control group, an ODN60rc-transfected group and an ODN60-transfected group, and cultured for 48 hours. For three rounds of transfection screening, the cells underwent transfection for 36 hours per round, with 36 hours rest intervals between rounds of transfection. The abundance of gRNAs for each gene in living cells was determined by deep sequencing. Screen results for the enrichment of genes (B) and the gRNAs targeting SLFN11 or TLR9 (C) are shown. Detail data can be found in data S2. ( D ) The effects of SLFN11 knockout on activation of the indicated pathway by CGT ODNs were analysed by immunoblotting. ( E to I ) Effects of SLFN11 knockout (E to H) or rescue expression (F) on cytokine expression induced by CGT ODNs (E and F), other types of nucleic acids (G), viral genome sequence-derived CGT ODNs (H) and the gDNA from E.coli , HIV1, and AAV2 (I). The indicated types of nucleic acids were transfected (E to H) or electroporated (I) into cells at a concentration of 1 μg/ml. ( J to L ), Immunofluorescence detection of ssDNA in WT and SLFN11 -/- HEK293 cells exposed to 4 mM HU (J), scale bar, 20 μm; mean fluorescence intensity of intracellular ssDNA per cells (K) and the mRNA level of TNF and CXCL8 (L) were measured. SLFN11 -/- HEK293 cells [(D to F) and (J to L)], SLFN11 -/- DU145 cells (G), and SLFN11 -/- 293A cells [(H) and (I)] were generated by CRISPRLJCas9. pcDNA3.1-SLFN11 was transfected for complementary expression (F). The results of qPCR assays [(E to I) and (L)] are expressed as the mean ± s.d. from three technical replicates, and the data are representative of three independent experiments (D to K).
Article Snippet: Canonical CpG ODNs including ODN1585, ODN1826 and ODN2395 were from a mouse TLR9 agonist kit (Invivogen, tlrl-kit9m).
Techniques: Genome Wide, CRISPR, Infection, Selection, Control, Transfection, Cell Culture, Sequencing, Knock-Out, Activation Assay, Western Blot, Expressing, Derivative Assay, Concentration Assay, Immunofluorescence, Fluorescence, Generated
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![( A and B ) Sonicated and untreated E. coli gDNA was denatured by heat (98 °C for 5 min) to generate ssDNA. The resulting gDNA with the indicated treatments was resolved on a 1% agarose gel with ethidium bromide (EB) staining (A) and assessed by SYBR Green melting curve analysis (B), shaded regions indicate standard error; NC, SYBR Green without gDNA. ( C ) The expression levels of genes in cGAS or <t>TLR9</t> signal pathway in the indicated cell types were analysed by RTLJPCR. The PCR products were resolved on a 1% agarose gel. ( D to F ) HEK293 cells were transfected with the indicated forms of E. coli gDNA. The ATP-based cell viability (D) and cell morphology (E) were examined 48 hours post-transfection, and TNF and CXCL8 mRNA levels were measured by qPCR (F) 12 hours post-transfection. ( G to I ) Trex1 protein levels in the indicated cell lines (G) and Trex1-reconstituted HEK293 cells (H) were determined by immunoblotting. TNF and CXCL8 mRNA levels (I) in WT and Trex-overexpressed (OE) HEK293 cells exposed to 4- or 8-mM HU were measured by qPCR. Data are shown as the mean ± s.d. from three technical replicates [(B), (E), (F), and (I)] and are representative of two [(A to C), (G), and (H)] or three [(D to F) and (I)] independent experiments.](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_93/10__1101_slash_2024__02__24__581893/10__1101_slash_2024__02__24__581893___F6.large.jpg)
![( A ) Transfection efficiency of top- and bottom-ranked ODNs. HEK293 cells were transfected with the indicated biotinylated ODNs and analysed as described in fig. S2A. ( B and C ) Morphology changes of HEK293 cells transfected with ODN60 containing the exchange of the indicated adjacent nucleotides. ( D ) ATP-based cell viability of HEK293 cells transfected with ODN60 with 3′ terminal truncations. ( E and F ) Single-nucleotide sensitivity of CGT/A motif for the ability of the top-ranked ODNs to induce cell morphology changes (E) and activate JNK pathways (F). ( G ) Positions of the CGT/A motifs in top-ranked and bottom-ranked ODNs in . ( H ) The percentage changes of four types of bases in CGT ODNs, relative to the stimulatory rank of . ( I ) Sequence comparison of CGT ODNs with canonical CpG ODNs regarded as TLR9 agonists. Capital letters, phosphodiester (PO) link; lowercase case, phosphorothioate (PS) link; underline, palindromic sequence. ( J and K ) Effects of PS linkage (J) and 5-methyl-deoxy-cytidine modification (K) on the stimulatory potency of the top-ranked ODNs. ( L ) WT and TLR9-knockout mouse lung fibroblasts were transfected with the indicated ODNs, and Ifnb1 mRNA expression was then measured by qPCR. HEK293 cells were transfected with the indicated ODNs at 120 mM, and phase-contrast images (scale bar, 50 μm) [(B), (C), and (E)] and ATP-based cell viability [(D), (J), and (K)] were examined 48 hours after transfection as representatives of immunostimulatory activity. Cell viability [(D), (J), and (K)] and qPCR (L) data are shown as the mean ± s.d. from three technical replicates. The data shown are representative of two (L) or three [(A to F), (J), and (K)] independent experiments.](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_93/10__1101_slash_2024__02__24__581893/10__1101_slash_2024__02__24__581893___F9.large.jpg)
![( A to C ) Schematic overview of genome-wide CRISPR screening for essential genes in response to ODN60 (A). HEK293 cells were infected with a Brunello lentiviral pool containing Cas9 and a gRNA library. After selection with puromycin, infected cells were divided into an untreated control group, an ODN60rc-transfected group and an ODN60-transfected group, and cultured for 48 hours. For three rounds of transfection screening, the cells underwent transfection for 36 hours per round, with 36 hours rest intervals between rounds of transfection. The abundance of gRNAs for each gene in living cells was determined by deep sequencing. Screen results for the enrichment of genes (B) and the gRNAs targeting SLFN11 or TLR9 (C) are shown. Detail data can be found in data S2. ( D ) The effects of SLFN11 knockout on activation of the indicated pathway by CGT ODNs were analysed by immunoblotting. ( E to I ) Effects of SLFN11 knockout (E to H) or rescue expression (F) on cytokine expression induced by CGT ODNs (E and F), other types of nucleic acids (G), viral genome sequence-derived CGT ODNs (H) and the gDNA from E.coli , HIV1, and AAV2 (I). The indicated types of nucleic acids were transfected (E to H) or electroporated (I) into cells at a concentration of 1 μg/ml. ( J to L ), Immunofluorescence detection of ssDNA in WT and SLFN11 -/- HEK293 cells exposed to 4 mM HU (J), scale bar, 20 μm; mean fluorescence intensity of intracellular ssDNA per cells (K) and the mRNA level of TNF and CXCL8 (L) were measured. SLFN11 -/- HEK293 cells [(D to F) and (J to L)], SLFN11 -/- DU145 cells (G), and SLFN11 -/- 293A cells [(H) and (I)] were generated by CRISPRLJCas9. pcDNA3.1-SLFN11 was transfected for complementary expression (F). The results of qPCR assays [(E to I) and (L)] are expressed as the mean ± s.d. from three technical replicates, and the data are representative of three independent experiments (D to K).](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_93/10__1101_slash_2024__02__24__581893/10__1101_slash_2024__02__24__581893___F3.large.jpg)