Abstract CRISPR-associated (Cas) transposases (CAST) are RNA-guided systems capable of giga notice horas 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, caravan scissor jack 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.