Fig. 4: Structural and functional consequences of PAM and target DNA recognition by PseQCascade.
From: Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells
a Top, overall view of the PseCAST QCascade complex, oriented to highlight the target DNA recognition. Bottom, Magnified view of the experimental cryoEM density map around Cas7.1 and Cas7.2, showing interactions with the crRNA (gray) and DNA target strand (TS, red). NTS, DNA non-target strand. b Magnified views of the PAM binding pocket, with Cas8 and DNA shown in blue and red, respectively. Residues A243 and A244 lack any base-specific, hydrogen-bonding interactions with the DNA. c Normalized DNA integration efficiency at the genomic AAVS1 locus in HEK293T cells for the indicated Cas8 mutants (top), plotted above the WebLogo for PAM preferences in the –1 and -2 positions (bottom) derived from integration into pTarget. (For additional PAM specificity data, see Supplementary Fig. 7e.) Integration efficiency data are shown as mean ± s.d. for n = 3 biologically independent samples. Source data are provided as a Source Data file. d Overlay of the refined atomic model and cryoEM density (semi-transparent) for the seed region of QCascade bound to the DNA target strand. e Schematic representation showing angles for the first five RNA-DNA base pairs (BP 1–5) within the R-loop. f View of the RNA-DNA heteroduplex at right, highlighting the unfavorable base-pairing surrounding flipped out nucleobases within the first 18 base pairs of the R-loop. g Magnified view of the RNA-DNA heteroduplex segments aligned at the flipped out base pair, revealing consistent unfavorable angles at the adjacent base pairs. h Normalized RNA-guided DNA integration efficiency at the genomic AAVS1 locus in HEK293T cells for the indicated Cas7 mutations, as measured by amplicon sequencing. Data are shown as mean ± s.d. for n = 3 biologically independent samples. Source data are provided as a Source Data file.
