E. coli strains, plasmids and the primed adaptation experiment
The E. coli KD263 strain is a derivative of BW40119 strain described earlier (28). It contains the cas3 gene under the control of the lac_UV5 promoter and the _casABCDE12 operon under the _ara_Bp8 promoter control. The KD263 strains harbors a single genetically modified CRISPR cassette with two repeats and a single g8 spacer described earlier (14). KD263 was transformed with a pG8_C1T plasmid, a derivative of the pT7blue cloning vector harboring a 209-bp fragment of the M13 bacteriophage DNA containing the g8 protospacer (28). The protospacer sequence harbors a C to T change at the position of +1 that renders CRISPR interference by the g8 spacer containing crRNA ineffective (14). KD263 cells transformed with pG8_C1T were grown overnight at 37°C in Luria-Bertani (LB) broth supplemented with 100 μg/ml ampicillin. Aliquots of the culture were diluted 200-fold into six individual tubes with fresh LB broth without ampicillin and supplemented with IPTG (isopropyl β-D-1 thiogalactopyranoside) and arabinose to the final concentration 1 mM each. The cultures were grown at 37°C overnight. The six individual cultures were mixed and genomic DNA was isolated from the pooled cultures. The cells were lyzed by 2-min incubation with 1 mg/ml lysozyme and DNA was purified by phenol, phenol/chloroform, chloroform extractions followed by ethanol precipitation. CRISPR expansion was monitored by polymerase chain reaction (PCR) in 20 μl reactions containing 20–50 ng genomic DNA with primers matched the CRISPR leader sequence and g8 spacer: Ec_LDR-F (5′-AAGGTTGGTGGGTTGTTTTTATGG-3′) and M13g8 (5′-GGATCGTCACCCTCAGCAGCG-3′) using Phusion High-Fidelity DNA Polymerase (New England Biolabs). Six independent amplification reactions were pooled, PCR products corresponding to expanded CRISPR cassette were gel purified using QIAquick Gel Extraction Kit (QIAGEN) and sequenced with MySeq Illumina System at Moscow State University Genomics facility as described (31).
Raw sequencing data were analyzed using ShortRead and BioStrings (34) packages. Illumina-sequencing reads were filtered for quality scores of ≥20 and reads containing two repeats (with up to two mismatches) were selected. Reads that contained 33-bp sequences between two CRISPR repeats were next selected. The 33-bp segments were considered spacers. Spacers were next mapped on the pG8_C1T plasmid with no mismatches allowed. R scripts and their package ggplot2 (35) were used for spacers statistics and graphical representation. Logo construction was done with http://weblogo.berkeley.edu (36).
During the process of prokaryotic CRISPR adaptation, a copy of a segment of foreign deoxyribonucleic acid referred to as protospacer is added to the CRISPR cassette and becomes a spacer. When a protospacer contains a neighboring target interference motif, the specific small CRISPR ribonucleic acid (crRNA) transcribed from expanded CRISPR cassette can protect a prokaryotic cell from virus infection or plasmid transformation and conjugation. We show that inEscherichia coli, a vast majority of plasmid protospacers generate spacers integrated in CRISPR cassette in two opposing orientations, leading to frequent appearance of complementary spacer pairs in a population of cells that underwent CRISPR adaptation. When a protospacer contains a spacer acquisition motif AAG, spacer orientation that generates functional protective crRNA is strongly preferred. All other protospacers give rise to spacers oriented in both ways at comparable frequencies. This phenomenon increases the repertoire of available spacers and should make it more likely that a protective crRNA is formed as a result of CRISPR adaptation.