Efficient encapsulation of CRISPR-Cas9 RNP in bioreducible nanogels and release in a cytosol-mimicking environment

Hydrogels are a promising delivery vehicle for many therapeutic modalities that can protect the embedded therapeutic during delivery and do not depend on time-consuming and expensive cell culture processes, such as those needed to make VLPs and AAVs. In this work, Westarp, et al. from the Beisel lab found that the myTXTL reaction mix can serve as a model reducing environment for a cytosol release assay of active Cas9 ribonucleoprotein complexes embedded in small hydrogels called nanogels. As the Cas9 RNPs are released from the nanogel into the myTXTL reaction, they are able to repress GFP expression from a plasmid target. This enables a convenient and rapid method for optimizing nanogel formulations and release kinetics that is not easily performed in cells.