Integration of therapeutic cargo into the human genome with programmable type V-K CAST
Jason Liu, Daniela S. Aliaga Goltsman, Lisa Alexander, Khak Khak Khayi, Jennifer Hong, Drew T. Dunham, Christine A. Romano, Morayma M. Temoche-Diaz, Shailaja Chadha, Rodrigo Fregoso Ocampo, Jennifer Oki, Owen P. Janson, Keirstinne Turcios, Liliana Gonzalez‐Osorio, Jared Muysson, Jenat Rahman, Sarah M. Laperriere, Audra E. Devoto, Cindy J. Castelle, Cristina N. Butterfield, Gregory J. Cost, Christopher T. Brown, Brian C. Thomas
Abstract
CRISPR-associated (Cas) transposases (CAST) are RNA-guided systems capable of programmable integration of large segments of DNA without creating double-strand breaks. Engineered Cascade CAST function in human cells but are challenging to deploy due to the complexity of the targeting components. Unlike Cascade, which require three Cas proteins, type V-K CAST require a single Cas12k effector for targeting. Here, we show that compact type V-K CAST from uncultivated microbes are repurposable for programmable DNA integration into the genome of human cells. Engineering for nuclear localization and function enables integration of a therapeutically relevant transgene at a safe-harbor site in multiple human cell types. Notably, off-targets are rare events reproducibly found in specific genomic regions. These CAST advancements are expected to accelerate applications of genome editing to therapeutic development, biotechnology, and synthetic biology. RNA-guided CRISPR-associated transposases (CAST) are natural systems with broad potential in biotechnology. Here, the authors report compact type V-K CAST discovered from genome-resolved metagenomics and demonstrate targeted integration of a large transgene to a safe-harbor site in the human genome.