After the successful proof of concept with an IVT system, we moved on to apply this blue ON and UV OFF system in CFPS (Fig. ). We incubated the bLA-mV DNA template in the CFPS system at 37 °C in the presence of uvLA-V3 and RNase H for 4 hours under different illumination conditions and measured intact protein yield by fluorescence (Fig. ). Illumination with only blue light showed an equal protein output compared to the no ASO control (p-value = 0.196), due to uvLA-V3 not activating under blue light. Irradiation with UV light alone did not activate the bLA-mV DNA template and no protein was produced after 4 hours, as only the ASO was activated. UV light was then applied at different timepoints, following blue light-activation of the template, to halt cell-free protein synthesis upon demand by degrading the mRNA already generated. When irradiated with UV at the same time as blue light, prior to incubation, a high degree of gene knockdown (74%) was observed, as any mRNA formed would be quickly degraded by RNase H and the uncaged ASO. When irradiating with UV following blue light-activation and 30 minutes incubation, protein synthesis had already initiated but was halted at 51%, compared to when no ASO was present. This is in line with previous data, showing that CFPS systems produce a substantial amount of mRNA and protein in the first stages of the reaction. Lastly, when irradiated with UV following blue lightactivation and 3 hours incubation, no reduction in protein output was observed (p-value = 0.519), as expected, because mVenus production already reached a plateau. This demonstrated a two wavelength ON and OFF switch for CFPS, with the ability to temporally activate and halt protein synthesis.