ENCOFRADO Y DESENCOFRADO

We next asked whether other components of the cytosolic sensing pathway were involved. IFI16 has been reported to signal through STING (Almine, 2017). IFI16 has been reported to signal through STING (Unterholzner, 2010; Almine, 2017) but has also been reported to have STING-independent functions in DNA damage and senescence (Xin, 2003). We therefore decided to test the involvement of STING in the innate immune response to DNA damage to determine if IFI16 was signalling through STING. To study the role of STING in

this response, we first used STING-targeting siRNA, to knock down STING levels in HaCaT

cells, and this was confirmed at the mRNA and protein level (Figure 16a, b). The STING gel

mobility shift observed in HT-DNA treated lanes is indicative of activation-induced STING phosphorylation (Tanaka, 2012). When compared with non-targeting siRNA treated cells,

STING knockdown cells had significantly reduced responses to Etoposide treatment as

measured by IFN-β mRNA (Figure 16c), and CCL20 mRNA (Figure 16e). The IL-6 mRNA

response in STING siRNA treated cells was decreased but not significantly so, this could be

an authentic phenotype or due to incomplete knockdown of STING protein (Figure 16d).

These results indicated that STING contributes to the Etoposide-induced innate immune

response. A slight decrease in H2AX phosphorylation was also observed in STING siRNA

treated cells (Figure 16a).

To validate the siRNA results, we then generated STING-null HaCaT cells. This was done

using CRISPR (Clustered regularly interspaced short palindromic repeat) Cas9 (CRISPR associated protein 9) technology. Cas9 is a DNA endonuclease which works, in prokaryotes, together with CRISPR sequences specific to pathogens, to defend against viral infection (Barrangou, 2007). This technology has been co-opted for use in genome editing in a wide variety of biological systems (Carroll, 2011). CRISPR works by transfecting a guide RNA targeting the gene of interest, as well as the Cas9 coding sequence. The Cas9 nuclease complexes with the guide RNAs which target the nuclease activity to cleave sequence specific DNA of the target gene (Gasiunas, 2012).

117 Figure 16: STING siRNA verification in HaCaTs

a, b. WT HaCaT cells were treated with Non-targeting (NT) siRNA or STING-targeting siRNA for 48 hours before treatment with DMSO control, 50μM Etoposide,

Lipofectamine, or 1μg/ml HT-DNA. Cells were then lysed and analysed for protein

expression by western blot (a) or STING mRNA expression by qRT-PCR (b).

c-e. Cells treated with siRNA as in (a) were then stimulated with DMSO or 50μM

Etoposide for 6 hours before lysis and quantification of IFN-β mRNA (c), IL-6 mRNA (d),

or CCL20 mRNA (e) by qRT-PCR.

Data are presented as mean values of biological triplicates. Error bars indicate standard deviations. N.s. = non-significant, p>0.05; ** p≤0.01, as determined by Student’s t-test. Data are representative of at least two experiments.

118

Wild type Cas9 nucleases produce double-stranded breaks in the target site, stimulating repair by either Non-Homologous End Joining, a faster but more error-prone mechanism, or Homologous Recombination. NHEJ can elicit a range of mutations or deletions at the site of the DNA break. The errors generated by NHEJ vary in size but even a small repair error can result in a frameshift mutation which is sufficient to be deleterious to the gene. This repair process can lead to the generation of cells deficient or mutated in the gene of interest (Figure 17). WT HaCaT cells were transfected with plasmids encoding Cas9, puromycin

resistance genes, and two guide RNAs targeting the start of STING exon 3. Transfected and

mock-transfected cells were then treated with puromycin over several days to eliminate cells which were not transfected. Selected cells were then left to recover for some days before they were seeded for clonal selection with approximately one cell per well in several 96-well plates. These clones were grown for several weeks or until wells appeared confluent. Plates were then split into three new plates; the first to continue growing clones, the second a PCR plate to test clones by HRM (High Resolution Melting), the third to be tested for protein

expression by western blot. This process is simplified in Figure 17. By HRM, the DNA of

each CRISPR clone was amplified around the site of the guide RNA. Any mutations in this sequence compared to WT cells should appear as a shift or an alteration in melt peaks (Ririe, 1997; Gundry, 2003). HRM increases the temperature of samples very slowly and precisely after PCR amplification under the two strands of amplicon DNA have separated. For two amplicons of the same sequence, the DNA should separate at the same

temperature. Mutations in the DNA can alter the temperature at which the DNA strands separate or “melt” and when this occurs, the dye bound to dsDNA stops fluorescing, a

process which is measurable. An example of these melt curves is shown in Figure 17c.

HRM candidates were selected which differ in melt curve appearance from WT samples. The corresponding clones were then lysed for analysis of target protein expression by western

blot (Figure 17d). Any clones which did not show expression of the target protein but did

express the loading control β-actin were then moved from 96-well plates into 6-well plates

and subsequently 25cm2 _{flasks. When sufficient cells were propagated, clones underwent a}

functional test, stimulation with transfected HT-DNA or lipofectamine control because

119 Figure 17: STING-/- _{cell generation}

a. Diagram of STING-targeting guide RNAs used in combination with Cas9 to generate

deleterious mutations in the STING gene in WT HaCaT cells.

b. Schematic showing the workflow used for Cas9 clone generation.

c. Examples of normalised melt peaks from HRM analysis of WT cells and STING

mutant candidates. DNA extracted from CRISPR-Cas9 transfected cell clones are amplified using primers specific to the guide RNA target site before high-resolution melting and analysis of melt curves to identify possible mutations.

d. An example of western blot screening of HRM candidates, further screened for STING

protein expression.

e. An example of qRT-PCR screening of candidates that test negative for protein

expression by western blot. WT and candidate clone HaCaT cells were treated with lipofectamine control (Lipo), or 1μg/ml HT-DNA (HT) for 6 hours before lysis for qRT-

120 Figure 18: STING-/- _{cell characterisation.}

a. Wild type HaCaT keratinocyte cells and 2 clonal HaCaT cell lines with a genetic deletion of

STING (STING-/-_{) were treated with 50μM Etoposide or DMSO control for 6 hours before lysis.}

Lysates were then analysed for protein expression by Western Blot.

b. WT and STING-/- _{HaCaTs seeded in single cell colonies and treated with 0μM, 0.1μM, or}

0.2μM of Etoposide for 10 days, were analysed for colony survival, by counting colonies that grew to >50 cells.

c. qRT-PCR analysis of p21/Waf1 mRNA expression in WT or STING-/- _{HaCaT cells 6h post}

treatment with 50µM Etoposide.

Data are presented as mean values of biological triplicates. Error bars indicate standard deviations. N.s. = non-significant, p>0.05 as determined by Students t-test. Data are

121

2009). These cells were compared to stimulated WT cells by qRT-PCR (Figure 17e). Cells

that passed this test were continued. Two STING-deficient cell clones were carried forward

and tested for expression of other DNA sensors and they were found to make a normal DNA damage response as seen by phosphorylation of H2AX upon damage, however this was

slightly reduced compared to WT cells (Figure 18a). This is similar to what was observed in

STING-targeting siRNA treated cells (Figure 16a). By clonogenic survival assay we found

that WT and STING-/- _{cells had no difference in cell colonies numbers after Etoposide}

treatment, indicating that they could repair themselves just as well as WT cells (Figure 18b).

STING-/- _{HaCaTs also showed activation of p21/Waf1 after Etoposide treatment, comparable}

to WT cells (Figure 18c).