Fig. 2: The role of crRNA in the PAM-distal region of PseQCascade.
From: Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells
a Overall view of the cryoEM reconstruction of the PseCAST QCascade complex. b Magnified view of the dashed region in (a), highlighting the cryoEM density (colored and semi-transparent) for interactions between the indicated crRNA nucleotides and protein subunits. c Magnified view of the dashed regions in (b), highlighting interactions between the crRNA and Cas6 (left), TniQ.1 (middle), and both TniQ.2 and Cas7.6 (right). Key interacting residues are labeled. d Normalized RNA-guided DNA integration efficiency at the genomic AAVS1 locus in HEK293T cells, as measured by amplicon sequencing. The indicated alanine mutations were designed to perturb specific RNA-protein interactions highlighted in (c), and were compared to WT. NT, non-targeting crRNA. Data are shown as mean ± s.d. for n = 3 biologically independent samples. Source data are provided as a Source Data file. e Comparison of the crRNA conformation within the PAM-distal region, adjacent to the site of RNA hairpin stabilization by Cas6, for VchCAST (PDB: 6PIJ) and PseCAST (this study). The region around nucleotide G41 exhibits a distinct configuration for PseCAST, likely affecting the behavior of the adjacent TniQ dimer.
