Figure 4.

Integration tolerance of varied 3′overhangs by Cas1-Cas2. A, plasmid integration assay showing variation in 3′ overhang tolerance between non-PAM strand (left) and PAM (strand). 3′-Phosphate used to block integration of the complementary strand is indicated by a red stop sign symbol containing the letter P. Reactions with varying overhang lengths (X) were run on an agarose gel alongside two control reactions: no prespacer (−PS) and prespacer with 5-nt overhangs containing 3′-phosphate blocks on both strands (5*). B, schematic of an oligonucleotide target integration assay showing reaction components and expected products of leader-side (61 nt) and spacer-side (113 nt) integration of the preprocessed prespacer containing 5-nt 3′ overhangs on both strands. C, denaturing sequencing gel of the oligonucleotide integration assay using prespacers with varied symmetric 3′ overhangs (X). Radiolabel is indicated with a star. Schematics of expected half-site products are shown on the right side of each gel. M, marker; minus or plus symbols indicate the absence or presence of the leader-repeat-spacer oligonucleotide shown in A. For clarity, zoomed-in cutouts of leader-side products, outlined on each gel, are shown below each gel.