Extended Data Fig. 7: PAM blocks Cas-mediated integration of foreign DNA into CRISPR repeat.
a, Schematic summarizing the step-wise strand-transfer reactions catalyzed by a Cas integrase. In the absence of putative host nucleases that trim PAMs, the foreign DNA fragments that contain a PAM stall the reaction at leader-side integration. However, the foreign DNA fragments that have been trimmed (no PAM) proceed through leader- and spacer-side integration. b-d, Endpoint integration reactions performed with a PAM-containing foreign DNA in triplicate, resolved on denaturing polyacrylamide gels. The X1 and X2 lanes signify lanes that were not further analyzed for this manuscript. e-g, Endpoint integration reactions performed with a trimmed foreign DNA in triplicate, resolved on denaturing polyacrylamide gels. The X1 and X2 lanes signify integration substrates that were not analyzed for this manuscript. h, i, Quantification of leader- (grey circles) or spacer-side (white circles) integration events from all three replicate gels. Individual dots for each triplicate reaction are shown, and some dots overlap. Three independent gels were run for PAM-containing or trimmed Foreign DNA integration reactions with similar results.
