In all panels, unexpanded CRISPR arrays are labeled UA; expanded arrays — EA, the leader is shown as a light peach rectangle; repeats — as white rectangles; the pre-existing spacer S⁰ — as a dark peach rectangle; the newly acquired spacer S⁺¹ — as a turquoise rectangle. Primers are shown as arrows below CRISPR arrays. A flipped end of a primer arrow indicates the absence of complementarity between the primer's end and the template. The brackets below primer pairs indicate PCR products (dashed lines correspond to low-efficiency PCR with degenerate primers whose 3′ ends are not complementary to the template). (A) Standard PCR-based spacer acquisition assay. The leader-proximal part of the CRISPR array is amplified with a leader-specific primer (light peach arrow) and a primer annealing to the S⁰ spacer (dark peach arrow). The products of PCR are resolved on an agarose gel. M, molecular-weight size marker. The band corresponding to expanded CRISPR arrays (EA) is purified from the gel and subjected to HTS (high-throughput sequencing). (B) Selective amplification of expanded arrays by PCR with a leader-specific primer and a degenerate repeat-specific primer (white arrows with turquoise arrowheads). All nucleotides of the degenerate primer, except for the last one, are annealed to the repeat placing the 3′-end nucleotide opposite the last nucleotide of the leader or the last nucleotide of the acquired spacer. The last position of the degenerate primer includes one of three nucleotides that are not complementary to the leader's last nucleotide enabling efficient amplification only when the last nucleotide of the acquired spacer is different from the last nucleotide of the leader. (C) Selective amplification of expanded arrays using CAPTURE. Top, CRISPR arrays are amplified and the band corresponding to expanded CRISPR arrays is purified after electrophoresis as in A. Bottom, to further increase the percentage of expanded arrays in sequencing libraries, the products of the first PCR are reamplified with primers (white arrows) annealing to repeats. The expanded arrays are selectively amplified since the products of the first-stage PCR of unexpanded and expanded arrays contain one and two repeats, correspondingly. (D) Selective amplification of expanded arrays using SENECA. An FaqI endonuclease recognition site (maroon rectangle) is introduced immediately following the first CRISPR repeat. FaqI cleaves DNA upstream of its recognition site creating sticky ends in the repeat sequence. A matching adapter is ligated to this sticky end and PCR amplification with adapter-specific (light blue) and repeat-matching (white) primers is performed.