Fig. 3: Type V-K CAST systems localize in human HEK293T cell nuclei.
From: Integration of therapeutic cargo into the human genome with programmable type V-K CAST
A Immunofluorescence of MG64-6 components localizing to the nucleus of human cells. Left: components tagged with an HA flag. Right: components tagged with a FLAG tag. Arrows indicate residual localization of TnsC in the cytoplasm, scale bars represent 10 µm. B Schematic representation for in vitro testing of nuclear extracts to determine the functionality of CAST components post-localization. Cells expressing components of interest are differentially lysed to yield cytoplasmic and nuclear extracts. Upon incubation with the addition of sgRNA, donor fragment, and pTarget, PCR junctions are amplified. C Integration junction PCR products of in vitro transposition reactions using nuclear extract inputs from cells expressing Cas12k-NLS with (lanes 3–6) and without (lanes 8–10) sso7d, NLS-TnsB, NLS-TnsC, and either NLS-HMGN1-TniQ or NLS-H1 core-TniQ. In vitro, control reactions contain all components expressed exclusively in vitro and tested in vitro. Expected bands for the forward (Fwd) left end (LE) and right end (RE) or reverse (Rev) LE and RE, with added sgRNA (+), indicate positive integration (arrows). D Genomic search space (bp) for single targets at high-copy loci in the human genome. The search space required to target the LINE-1a 3’ (10,000 copies), SVA (1000 copies), Mariner (50 copies), HERV (4 copies), or a unique human target (1) are compared to the search space required to target a unique E. coli site. E In vitro integration junction PCR products at high-copy targets in purified human genomic DNA. Expected bands for the Fwd LE or Rev LE rows indicate positive integration (arrows). Experiments in A, C, E were independently replicated twice.
