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 Drying Racks 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 Machine Accessories 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.