Gene expression in cell-free systems has previously been controlled with light, by either chemically introducing light-activated photocages into the DNA templates for transcription, or by using light-sensitive proteins. The majority of these systems have leaky 'off-states', where expression still occurs without light, and in the case of light-sensitive proteins require the expression of additional genes, both of which limit their application. We have previously developed an approach for the light-activation of cell-free protein synthesis (CFPS) by attaching photocaged biotin/monovalent streptavidin (mSA) to a T7 promoter sequence to sterically block transcription, prior to illumination . Photocleavable biotin linkers were attached to amino-modified oligonucleotides and used as primers to amplify any gene of interest for control using light. The advantage of this approach is that it is made from commercially available modified nucleic acids and photocleavable biotin, and has a tight 'offstate', with negligible expression without illumination. We have used this light-activated DNA (LA-DNA) to spatiotemporally activate protein synthesis in synthetic tissues, activate communication between synthetic cells and living cells, and, by employing orthogonal photocages, to engineer a dual wavelength cell-free AND-gate. The main disadvantage of our current approach is that it is an irreversible ON switch. Azobenzene-modified DNA promoters have been used as photoreversible switches; however, they are leaky in the 'off-state' and do not degrade the RNA already generated. In this project, we have applied our simple photocleavable biotin/streptavidin approach to develop lightcontrolled CFPS OFF switches from antisense oligonucleotides (ASOs) (Fig. ). ASOs are short DNA sequences that can selectively degrade a target mRNA in the presence of Ribonuclease H (RNase H), a common endonuclease. While light-activated ASOs have previously been generated, they are difficult to synthesise and orthogonally controlled versions have never been realised. The use of ASOs in cell-free systems has been greatly underexplored; however, 'transfection-style' methods have been developed to explore their use in synthetic cells. Here, we attached orthogonal UV (nitrobenzyl) and blue (coumarin) photocaged biotins, and then monovalent streptavidins, to amino-C6-dTs positioned across ASOs to tightly control gene knockdown in cell-free systems using light (Fig. ). We demonstrated the orthogonal degradation of two different mRNAs, depending on the wavelength used, and combined UV-controlled ASOs with our previously generated blue lightactivated DNA templates to precisely remote-control CFPS (Fig. ).