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Figure 1.

Detection of acquisition of interference-proficient spacers (S+1) in bacteriophage insensitive mutants (A) or in cells that lost plasmids (B). The structures of unexpanded (UA) CRISPR arrays in cells from an initial culture or of expanded (EA) arrays in cells that either survived phage infection or lost the plasmid are shown at the top. The leader (light peach rectangle), CRISPR repeats (white rectangles), pre-existing spacer S0 (dark peach rectangle), and newly acquired spacer S+1 (turquoise rectangle) are shown. Acquisition of spacers is tested by PCR with primers matching the leader (light peach arrow), and the S0 spacer (dark peach arrow). Amplicons from expanded and unexpanded arrays are shown as brackets below the primers. In A, a colony formed after phage infection is directly tested by PCR and results of agarose gel electrophoresis of amplicons obtained with starting cells (1) and the phage-resistant colony (2) are schematically shown on the right. In B, a liquid culture is inoculated with cells from an antibiotic-resistant (Ab-resistant) plasmid-bearing colony (1) with an unexpanded CRISPR array. After growth and plating on non-selective (−Ab) medium, the presence of the plasmid is tested by streaking colonies (2, 3, 4) on plates with and without Ab. Cells from the antibiotic-sensitive colony (2) are further tested by PCR to reveal a spacer acquisition event. M, molecular-weight size marker. Detection of CRISPR adaptation using cat (C) or yfp (D) reporter systems. Insertion of an inverted leader/single-spacer CRISPR array disrupts the reading frame of cat (C) or yfp (D) reporter genes. The elements of CRISPR arrays are shown as in A and B. Preporter — a promoter directing the synthesis of reporter mRNA; PCRISPR — a promoter directing the synthesis of pre-crRNA. The composition of reporter mRNAs is shown below. Each rectangle corresponds to one codon; rbs — a ribosome binding site; AUG — a start codon, UAA — a stop codon located in-frame with the AUG. Translation of mRNAs transcribed from unexpanded CRISPR reporters stops at the UAA codon in the leader sequence resulting in (C) chloramphenicol-sensitivity (Cm-sensitivity) or (D) absence of fluorescence. Insertion of a 61-bp (33-bp spacer/28-bp repeat) unit into the CRISPR array shifts the in-leader UAA stop codon out of the frame and restores the reporter gene reading frame resulting in chloramphenicol-resistance (C) or fluorescence (D) of cells with expanded CRISPR arrays.

Selection-based methods to detect CRISPR adaptation.

Detection of acquisition of interference-proficient spacers (S⁺¹) in bacteriophage insensitive mutants (A) or in cells that lost plasmids (B). The structures of unexpanded (UA) CRISPR arrays in cells from an initial culture or of expanded (EA) arrays in cells that either survived phage infection or lost the plasmid are shown at the top. The leader (light peach rectangle), CRISPR repeats (white rectangles), pre-existing spacer S⁰ (dark peach rectangle), and newly acquired spacer S⁺¹ (turquoise rectangle) are shown. Acquisition of spacers is tested by PCR with primers matching the leader (light peach arrow), and the S⁰ spacer (dark peach arrow). Amplicons from expanded and unexpanded arrays are shown as brackets below the primers. In A, a colony formed after phage infection is directly tested by PCR and results of agarose gel electrophoresis of amplicons obtained with starting cells (1) and the phage-resistant colony (2) are schematically shown on the right. In B, a liquid culture is inoculated with cells from an antibiotic-resistant (Ab-resistant) plasmid-bearing colony (1) with an unexpanded CRISPR array. After growth and plating on non-selective (−Ab) medium, the presence of the plasmid is tested by streaking colonies (2, 3, 4) on plates with and without Ab. Cells from the antibiotic-sensitive colony (2) are further tested by PCR to reveal a spacer acquisition event. M, molecular-weight size marker. Detection of CRISPR adaptation using cat (C) or yfp (D) reporter systems. Insertion of an inverted leader/single-spacer CRISPR array disrupts the reading frame of cat (C) or yfp (D) reporter genes. The elements of CRISPR arrays are shown as in A and B. P_reporter — a promoter directing the synthesis of reporter mRNA; P_CRISPR — a promoter directing the synthesis of pre-crRNA. The composition of reporter mRNAs is shown below. Each rectangle corresponds to one codon; rbs — a ribosome binding site; AUG — a start codon, UAA — a stop codon located in-frame with the AUG. Translation of mRNAs transcribed from unexpanded CRISPR reporters stops at the UAA codon in the leader sequence resulting in (C) chloramphenicol-sensitivity (Cm-sensitivity) or (D) absence of fluorescence. Insertion of a 61-bp (33-bp spacer/28-bp repeat) unit into the CRISPR array shifts the in-leader UAA stop codon out of the frame and restores the reporter gene reading frame resulting in chloramphenicol-resistance (C) or fluorescence (D) of cells with expanded CRISPR arrays.

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