Fig. 2: Protein trans-splicing rAAV vectors for in vivo delivery of split-CRISPR components.
From: Therapeutic in vivo genome editing: innovations and challenges in rAAV vector-based CRISPR delivery
A Schematic diagram of the protein trans-splicing rAAV-CRISPR system, in which split-inteins mediate the reconstitution of full-length proteins from N-terminal (NT) and C-terminal (CT) precursors of the Cas effector. B Trans-splicing rAAV-CBE vector system utilizing split nickase Cas9 (nCas9), rAPOBEC1 deaminase, and split inteins for cytidine base editing. nCas9NT is fused to rAPOBEC1 and the N-terminal fragment of the DnaB split-intein (Rma-N) derived from Rhodothermus marinus, whereas nCas9CT is fused to uracil-DNA glycosylase inhibitor (UGI), and the C-terminal fragment of the Intein (Rma-C). C Trans-splicing rAAV-ABE vectors encoding split nCas9, TadA deaminase, and N- and C-terminal inteins for reconstitution of the full-length ABE. nCas9NT is fused to the TadA/TadA* heterodimer and the N-terminal fragment of DnaE intein derived from Nostoc punctiforme (Npu-N), while nCas9CT is fused to the corresponding C-terminal fragment (Npu-C). D Trans-splicing rAAV-PE vector system encoding split nCas9, Moloney murine leukemia virus reverse transcriptase (MMLV-RT), pegRNA, and gRNA for efficient prime editing. All vector schematics indicate the positions of inverted terminal repeats (ITRs), promoters (CAG, CMV, Cbh, and U6), polyadenylation signals (pA), nuclear localization signals (NLS), epitope tags (V5 and HA), and other regulatory elements.
