Figure 9. Design and Synthesis of Cas-Reponsive Hydrogel Networks.

Methacryl-functionalized DNA is incorporated into polyacrylamide chains (PA-X, PA-Y) during starting macromer polymerization. This enables different routes to gel actuation and response modes. In one example, shown on the left, the Cas12a–gRNA is added to the gel precursor with the nanoparticle cargo, before the addition of dsDNA cues and ssDNA crosslinker. In another example, shown on the right, cell encapsulation is triggered through the addition of a small amount of ssDNA bridge crosslinker to the macromers mixed in solution. This thickens the pre-gel solution and minimizes losses incurred during the washing step. More ssDNA linker is then added at the same time as the cells to fully crosslink the hydrogels. Finally, the experiment is initiated by exposing the gels to gRNA-complexed Cas12a and dsDNA. Additional details of the crosslinking strategy (bottom of the panel): the two ends of the DNA bridge hybridize with distinct ssDNA anchors incorporated into polyacrylamide macromers, while the central AT-rich portion remains single-stranded and sensitive to Cas12a collateral activity. Reproduced with permission from Gayet et al.¹²⁷ Copyright 2020, Springer Nature.