Supplementary Information

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1 Supplementary Information Supplementary Figure 1: Synthetic annealed poly(ra):poly(dt) hybrid induces type I interferon response. a) Bone marrow derived macrophages (BMDM) were transfected with or without poly(ra), poly(dt) or poly(ra):poly(dt) for 6 hrs. RT-PCR was performed to check for the expression of Ifn-α, Ifn-β, Il-6 and Ip-1. b) Differentiated Human monocyte THP-1 cells were transfected with different amounts of poly(ra):poly(dt) (hybrid) or poly(da):poly(dt) dsdna and IP-1 in supernatants was measured by ELISA. In all figures error bars represent SEM. Supplementary Figure 2: Scheme representing the coding region of IFIT1, MAVS, STING and cgas targeted by the CRISPR/Cas9 system Depicted is a schematic representation of the genomic loci of human IFIT1, MAVS, STING and cgas genes. Small black and large grey squares represent non-coding and coding exons, respectively. The red arrow highlights the location of the CRISPR target site. The sequences under the genomic loci are a magnification of the targeted region. The top sequences are the reference sequences, whereby the red letters highlight the protospacer (CRISPR binding site) and orange letters the PAM (protospacer adjacent motif). For MAVS, STING and cgas the sequences below the reference sequence show the mutations that were detected by deep sequencing. Supplementary Figure 3: cgas-dependent secretion of IL-6 WT THP-1 cells or cgas KO THP-1 cells were transfected with or without poly(ra):poly(dt) (hybrid), pdna or ppprna for 16hrs and amount of IL-6 secreted was detected by ELISA. Supplementary Figure 4: Single stranded homopolymeric polynucleotides do not activate cgas a) RP-HPLC chromatograms showing the synthesis of cgamp(2-5 ) after incubation of recombinant cgas protein for 9 minutes in the presence of! 1!

2 ATP and GTP with different ligands (i) with poly(ra), (ii) with poly(dt), (iii) poly(da) and (iv) poly(ru). b) Synthetic dsdna, dsrna and RNA:DNA hybrids were incubated for 45, 9 or 18 minutes with recombinant cgas and the synthesis of 2 5 cgamp checked by RP-HPLC. In order to normalize for the total amount of sample loaded the % of AUC was calculated for each time point and each sample. Supplementary Figure 5: cgas activation loop upon dsdna and RNA:DNA hybrid binding Structure of cgas bound to dsdna (a) and RNA:DNA hybrid in two modes (b) and (c). The DNA strand is shown in green, whereas the RNA strand is depicted in light pink. The activation loop of cgas is highlighted in blue. Supplementary Figure 6: Induction of Type I IFNs by poly(dt) is cgas dependent. WT THP-1 cells or cgas KO THP-1 cells were transfected with or without poly(ra), poly(dt) and poly(ra):poly(dt) (hybrid) for 16hrs and amount of IP-1 secreted was detected by ELISA.!! 2!

3 Supplementary Figure 1 A Fold change over Hprt Ifn BMDM Il Ip-1 _ poly(ra) poly(dt) poly(ra) :poly(dt) pdna B 3 IP-1 (ng/ml) 2 1 THP-1 poly(ra) :poly(dt) poly(da) :poly(dt) (ng)

4 Supplementary Figure 2 A human IFIT1 genomic locus CAGAGATCGCATACCCAGCGCTGGATTCAGGGTTTTCAGGGATAGCGGACCTACCGAAATTTGGAAGCAAAGAAAATGGCTTGTGATTTTTTGTGGCTAA CTGAAAACCCTGAATCCAGCGCTGGGTATGCGATCTCTGCCTATCGCCTGGATGGCTTTAAATTAGCCACAAAAAATCACAAGCCATTTTCTTTGCTTCC homology arm 2A Gaussia luciferase gene homology arm B human MAVS genomic locus Exon3 TGGGTGGAGTACTTCATTGCGGCACTGAGGGGCTGTGAGCTAGTTGATCTCGCGGACGAAGTGGCCTCT TGGGTGGAGTACTTCATTGCGGCACTG TGAGCTAGTTGATCTCGCGGACGAAGTGGCCTCT REF -Del C human STING genomic locus Exon3 ATGCCCCACTCCAGCCTGCATCCATCCATCCCGTGTCCCAGGGGTCACGGGGCCCAGAAGGCAGCCTTGG REF ATGCCCCACTCCAGCCTGCATCCATCCATCCCGTGG-----GGGTCACGGGGCCCAGAAGGCAGCCTTGG -5nt ATGCCCCACTCCAGCCTGCATCCATCCATCCCGTGTCCA-GGGGTCACGGGGCCCAGAAGGCAGCCTTGG -1nt D Exon1 human cgas genomic locus CGACTTCCTGGCGGGGCCGAACTTTCCCGCCTTAGGCAGGGCGGCCTCGGGGGCAGCCGGAGACTCGGTG REF CGACTTCCTGGCGGGGCCGAACTTTCCCGCCTT-GGCAGGGCGGCCTCGGGGGCAGCCGGAGACTCGGTG -1nt CGACTTCCTGGCGGGGCCGAACTTTCCCGCCTTAG-CAGGGCGGCCTCGGGGGCAGCCGGAGACTCGGTG -1nt

5 Supplementary Figure 3 IL-6 (pg/ml) poly(ra):poly(dt) pdna ppprna WT THP-1 cgas KO - - +

6 Supplementary Figure 4 A 4.e-2 (i) poly(ra) 4.e-2 (ii) poly(dt) AU 3.e-2 2.e-2 AU 3.e-2 2.e-2 1.e-2 1.e Time Time e-2 (iii) poly(da) 4.e-2 (iv) poly(ru) AU 3.e-2 2.e-2 AU 3.e-2 2.e-2 1.e-2 1.e Time Time B 6 % AUC 4 2 poly(da):poly(ru) (dsrna) poly(ra):poly(dt) (Hybrid) poly(da):poly(dt) (dsdna) (mins)

7 Supplementary Figure 5 A B x-ray structure cgas + dsdna C docking model cgas + DNA : RNA hybrid docking model cgas + DNA : RNA hybrid

8 Supplementary Figure 6 8 IP-1 (pg/ml) poly(ra) poly(dt) poly(ra):poly(dt) WT THP-1 cgas KO - - +