Fig. 1: Type V-K CAST are active systems for programmable integration.
From: Integration of therapeutic cargo into the human genome with programmable type V-K CAST
A Type V-K CAST MG64-1 genomic context. The transposon is characterized by terminal inverted repeats (TIR, light salmon bars) that define the transposon left end (LE) and right end (RE), transposon genes TnsB, TnsC, and TniQ, the dead effector Cas12k (orange arrow), a tracrRNA (pink arrow), and CRISPR array. A predicted “TAAA” target site duplication (dark blue bars flanking the transposon’s TIRs), a pseudo repeat (trimmed wine-colored bar), and a self-targeting spacer (teal bar) were also identified. B Unrooted phylogenetic tree of Cas12k effector sequences. Cas12k effectors recovered here are shown as orange (confirmed transposon features) and black branches (only Cas effector), while reference Cas12k effector sequences are shown in gray. Reference sequences ShCas12k and AcCas12k are highlighted with red arrows. C Active single guide RNA design for MG64-1 and MG64-6. D Schematic of integration products assayed in in vitro experiments. Four junction PCR products are expected based on the orientation of integration: forward (Fwd) or reverse (Rev). E Integration junction PCR products for CAST systems MG64-1 (left lanes) and MG64-6 (right lanes) in vitro. Experiments were independently replicated twice. Product labels are derived from the schematic in (D). F SeqLogo representations of determined PAMs for MG64-1 (left) and MG64-6 (right).
