Materials and Methods

Sequences spanning the leader-repeat junction mediate CRISPR adaptation to phage inStreptococcus thermophilus

MATERIALS AND METHODSStrains and plasmidsStreptococcus thermophilus DGCC7710 (Sth) and phage 2972 were kindly provided by Dr. Sylvain Moineau. Sth was maintained in M17 medium (Oxoid) supplemented with 0.5% lactose (LM17) (3). Sth cultures were grown at 37° for overnight, 42° during the day and 30° for strains harboring temperature sensitive plasmids derived from pINTRS (57). E. coli Top10 was used for cloning and plasmid maintenance. When needed, erythromycin was supplemented at 150 μg/ml and 15 μg/ml for E. coli and Sth, respectively. Strains used in this study are listed in Supplemental Table S1.DNA manipulationWe followed standard procedures for cloning. Phusion polymerase, restriction enzymes and T4 DNA ligase from New England Biolabs (NEB) were used for cloning. Taq polymerase (with crimson taq buffer) (NEB) was used for colony polymerase chain reaction (PCR). Zymoclean™ Gel DNA Recovery Kit (Zymo Research) was used for gel extraction. QIAprep Spin Miniprep Kit (Qiagene) was used for plasmid preparations.Construction of CRISPR1 variantsThe methods developed by Renye and Somkuti using plasmid pINTRS were used to construct CRISPR1 variants on the Sth chromosome (57). Plasmid pINTRS contains upstream and downstream homologous regions of a pseudogene locus that encodes truncated components of the glucose phosphoenolpyruvate-dependent phosphotransferase system (PTS locus) (57). Constructs inserted into PTS locus (Figures ​(Figures44 and ​and5)5) were first cloned into pINTRS. Plasmids with correct inserts were confirmed by sequencing and transformed into Sth via electroporation (40). Transformants harboring desired plasmids were first grown at 30° under selection (15 μg/ml erythromycin). Plasmid integration into the genome was selected by shifting the growth temperature to 37° in the presence of erythromycin for 8 h and subsequently plated on LM17 (with 1% agar) to achieve single colonies. Colonies with plasmid integrated were grown in LM17 broth at 37° with selection. Excision of the plasmid and plasmid loss were allowed to occur by shifting the growth temperature to 30° without selection for 4–5 days with two subcultures (1:500 dilution) per day. Cells that had lost the plasmid were identified through patching of single colonies on LM17 plates with and without selection. Correct mutations were confirmed via PCR amplification and sequencing of the PTS region.Open in a separate windowFigure 4.Leader sequence directs adaptation. (A) A minimal CRISPR1 comprised of the leader (157 bp upstream of the repeat) and a repeat sequence (‘R’ black) was introduced at an ectopic locus in Sth. The promoter region and transcribed region of the leader are indicated in blue and green, respectively. A right arrow and ‘+1’ indicate the transcription start site. (B) Variants of CRISPR1 were engineered at the ectopic site with various sequences inserted between the spacer and second repeat of an L-R-S-R locus as indicated. The inserts are: 32 bp transcribed leader region (green, denoted ‘L32’) in ‘L-R-L32-R’, mutated transcribed leader region (gray, denoted ‘Lmut32’) in ‘L-R-Lmut32-R’ and +1 to +32 of the transcribed region of pSTH2201 (purple, denoted ‘Lp2201’) in ‘L-R-Lp2201-R’. Experimentally observed adaptation events are indicated at the downward arrows at each site as in Figure ​Figure22.Open in a separate windowFigure 5.Adaptation does not depend on specific leader promoter sequences. Variants of CRISPR1 were engineered at the ectopic site with various substitutions of the promoter and transcribed regions of the leader of an L-R-S-R locus as indicated. The leaders of ‘pCr3-L32-R-S-R’ and ‘pCr3-R-S-R’ contain the promoter region of CRISPR3 (light blue) and the transcribed region of either CRISPR1 (green) or CRISPR3 (dark blue), respectively as indicated. The leaders of ‘p2201-L32-R-S-R’ and ‘p2201-R-S-R’ contain the promoter region of pSTH2201 (light purple) and the transcribed region of either CRISPR1 (green) or pSTH2201 (dark purple), respectively as indicated. Sequences of the transcribed regions are shown. Experimentally observed adaptation events are indicated at the downward arrows at each site as in Figure ​Figure22.CRISPR1 variants at the native locus were similarly constructed (Figures ​(Figures2,2, ​,66 and ​and7),7), with a few alterations. We first constructed a derivative of pINTRS by replacing the PTS homologous regions with a multiple cloning site, yielding plasmid pINTRS-MCS. Seven hundred to eight hundred upstream and downstream homologous regions of CRISPR1 locus were PCR amplified, combined to a single fragment by overlap PCR, cloned into pINTRS-MCS and sequenced to yield pINTRS-Cr1. Mutations of the CRISPR1 leader and the repeat were achieved via quikchange mutagenesis on pINTRS-Cr1. The above protocol for chromosomal insertions into PTS locus was followed to accomplish deletions or mutations at the native CRISPR1 locus.Open in a separate windowFigure 2.The leader and a single repeat are required for adaptation. Adaptation was examined at various truncated CRISPR1 loci as described in Figure ​Figure1.1. ‘L-R-S-R’ retains the first spacer (‘S’, red) with upstream and downstream repeats (‘R’, black). ‘L-R’ includes a single repeat. The leader is divided into promoter region (blue) and transcribed region (green), with transcription start site indicated with a right arrow and ‘+1’. Downward arrow indicates the leader-repeat junction where new spacer acquisition generally occurs. Occurrence of adaptation (detected by PCR in multiple independent experiments) is indicated by ‘Yes’ or ‘No’. Numbers of adaptation events observed among total survivors examined (by PCR in independent experiments) are indicated.Open in a separate windowFigure 6.Leader sequences essential for adaptation are found in 10 bp region adjacent to repeat. (A) Three segments of the 32 bp transcribed region of the CRISPR1 leader (green) were mutated in the context of the L-R-S-R locus (Figure ​(Figure2).2). Substitutions (adenine<−>guanine, cytosine<−>thymine) were made individually in the repeat-proximal 10 bp (gray in ‘Lmut1-R-S-R’), central 10 bp (gray in ‘Lmut2-R-S-R’) and repeat-distal 12 bp (gray in ‘Lmut3-R-S-R’) of the transcribed region. Sequences of the transcribed regions are shown. (B) The ‘L10-R-S-R’ locus includes just the repeat-proximal 10 bp of the CRISPR1 leader transcribed region (green); the first two segments (22 bp) of the transcribed region are deleted (strikethrough). Experimentally observed adaptation events are indicated at the downward arrows at each site as in Figure ​Figure22.Open in a separate windowFigure 7.Repeat sequence adjacent to the leader is critical for adaptation. Constructs with mutations in the repeat element of the minimal CRISPR1 locus L-R (Figure ​(Figure2)2) were examined. One or two nucleotides at the leader-proximal end (‘L-Rmut1’ and ‘L-Rmut2’) or at the leader-distal end (‘L-Rmut3’ and ‘L-Rmut4’) of the repeat were substituted, respectively as indicated. Sequences of the repeats are shown. Experimentally observed adaptation events are indicated at the downward arrows at each site as in Figure ​Figure22.Phage infection and adaptation analysisOvernight cultures of Sth strains were diluted into fresh media (1:100) and grown for 2–3 h at 42° until the optical densities (A600) reached ∼0.3. Phage infection was performed with phage 2972 at multiplicity of infection of 0.3 (3,40). Survivors were tested for spacer acquisition by using specific primers (listed in Supplemental Table S2) for the CRISPR locus of interest.

Article TitleSequences spanning the leader-repeat junction mediate CRISPR adaptation to phage inStreptococcus thermophilus

Abstract

CRISPR-Cas systems are RNA-based immune systems that protect prokaryotes from invaders such as phages and plasmids. In adaptation, the initial phase of the immune response, short foreign DNA fragments are captured and integrated into host CRISPR loci to provide heritable defense against encountered foreign nucleic acids. Each CRISPR contains a ∼100–500 bp leader element that typically includes a transcription promoter, followed by an array of captured ∼35 bp sequences (spacers) sandwiched between copies of an identical ∼35 bp direct repeat sequence. New spacers are added immediately downstream of the leader. Here, we have analyzed adaptation to phage infection inStreptococcus thermophilusat the CRISPR1 locus to identifycis-acting elements essential for the process. We show that the leader and a single repeat of the CRISPR locus are sufficient for adaptation in this system. Moreover, we identified a leader sequence element capable of stimulating adaptation at a dormant repeat. We found that sequences within 10 bp of the site of integration, in both the leader and repeat of the CRISPR, are required for the process. Our results indicate that information at the CRISPR leader-repeat junction is critical for adaptation in this Type II-A system and likely other CRISPR-Cas systems.


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