Integration of therapeutic cargo into the human genome with programmable type V-K CAST

Jason Liu, Daniela S. Aliaga Goltsman, Lisa M. Alexander, Khak Khak Khayi, Jennifer H. Hong, Drew T. Dunham, Christine A. Romano, Morayma M. Temoche-Diaz, Shailaja Chadha, Rodrigo Fregoso Ocampo, Jennifer Oki-O’Connell, 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 () and Brian C. Thomas
Additional contact information
Jason Liu: Inc
Daniela S. Aliaga Goltsman: Inc
Lisa M. Alexander: Inc
Khak Khak Khayi: Inc
Jennifer H. Hong: Inc
Drew T. Dunham: Inc
Christine A. Romano: Inc
Morayma M. Temoche-Diaz: Inc
Shailaja Chadha: Inc
Rodrigo Fregoso Ocampo: Inc
Jennifer Oki-O’Connell: Inc
Owen P. Janson: Inc
Keirstinne Turcios: Inc
Liliana Gonzalez-Osorio: Inc
Jared Muysson: Inc
Jenat Rahman: Inc
Sarah M. Laperriere: Inc
Audra E. Devoto: Inc
Cindy J. Castelle: Inc
Cristina N. Butterfield: Inc
Gregory J. Cost: Inc
Christopher T. Brown: Inc
Brian C. Thomas: Inc

Nature Communications, 2025, vol. 16, issue 1, 1-14

Abstract: 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.

Date: 2025
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DOI: 10.1038/s41467-025-57416-2

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