(A) Overview of the chip-hybridized affinity-mapping platform (CHAMP) workflow. DNA is regenerated on a sequenced NGS chip. A subset of clusters is hybridized to fluorescent oligonucleotides (alignment markers, magenta). Fluorescent proteins are incubated in the chip (green) and the fluorescent intensities at each DNA cluster are recorded via TIRF microscopy. A computational pipeline uses the alignment markers to identify the DNA sequences of all fluorescent clusters.

(B) A schematic representation of the T. fusca Cascade protein complex. Cse1 is shown in purple, Cas7 subunits are shown in alternating blue and yellow, and all other subunits are collectively represented in gray. The target DNA is gray, the protospacer adjacent motif (PAM) and seed regions are black, while the crRNA is red.

(C and D) Increasing concentrations of fluorescent Cascade complexes are incubated in the regenerated NGS chip (C) and (D) the apparent binding affinities for each DNA sequence are obtained by fitting the fluorescent intensities to the Hill equation. The lowest-affinity curve in (black dashed line, D) reports non-specific binding of Cascade to off-target DNA clusters.

(E) Illustration of the synthetic oligonucleotide library used for CHAMP.

(F) Overview of the randomized library used for these studies. The bar graph represents the number of unique sequences used in the CHAMP experiments with increasing substitutions from the ideal PAM and protospacer sequence. The bars are shaded to indicate the percent coverage of the relevant sequence space. Violin plots indicate the number of DNA clusters observed per sequence in the CHAMP dataset. Only sequences represented by five or more unique DNA clusters are included in the analysis (dashed line).

(G) CHAMP experiments were highly repeatable between two independently sequenced NGS chips. The gray zones indicate ABAs that fell outside of our experimentally defined cutoff for non-specific binding. The r value was calculated omitting gray zones.