Materials and Methods

Conserved motifs in the CRISPR leader sequence control spacer acquisition levels in Type I-D CRISPR-Cas systems

MATERIAL AND METHODSBacterial strains and growth conditions E. coli DH5α and BW25113 strains were grown in Lysogeny Broth (LB) at 37ºC and continuous shaking at 180 rpm or grown on LB agar plates (LBA) containing 1.5% (wt/vol) agar. When required, the media were supplemented with 100 µg ml–1 ampicillin and 25 µg ml–1 chloramphenicol (see Table S1 (Supporting Information) for plasmids and corresponding selection markers).Plasmid construction and transformationPlasmids used in this study are listed in Table S1 (Supporting Information) . All cloning steps were performed in E. coli DH5α. Primers described in Table S2 (Supporting Information) were used for PCR amplification of the type I-D CRISPR locus (leader-repeat-spacer1) from Synechocystis sp. 6803 cell material using the Q5 high-fidelity Polymerase (New England Biolabs). PCR amplicons were subsequently cloned into the pACYCDuet-1 vector system (Novagen (EMD Millipore) using restriction-ligation cloning. The pCRISPR leader mutants were obtained by PCR-based mutagenesis using primers listed in Table S2 (Supporting Information) . All plasmids were verified by Sanger-sequencing (Macrogen Europe, Amsterdam, The Netherlands). Bacterial transformations were either carried out by electroporation (200 Ω, 25 μF, 2.5 kV) using a ECM 630 electroporator (BTX Harvard Apparatus) or using chemically competent cells prepared according to manufacturer's manual (Mix&Go, Zymo research). Electrocompetent cells were prepared following a protocol adapted from (Gonzales et al. 2013). Transformants were selected on LBA supplemented with appropriate antibiotics.In vivo spacer acquisition assay E. coli BW25113 was transformed with pCas1–2 and pCRISPR with varying lengths of the leader sequence (Table S1, Supporting Information). Cultures were inoculated from single colonies and passaged once after 24 hours of growth at 37˚C and continuous shaking at 180 rpm. 200 µL of cells cultured for 48 hours were harvested by centrifugation and resuspended in 50 µL of MilliQ water. Subsequently, 2 µL of cell suspension was subjected to spacer detection PCR using a forward primer annealing in the 3’ end of the CRISPR repeat of pCRISPR but mismatching the first nucleotide of spacer 1 (degenerated primer mix, BN143 + BN144 + BN145) (Heler et al. 2015) and a reverse primer annealing in the vector backbone (BN172) (Table S2, Supporting Information). PCR products were separated on 2% agarose gels and were densitometrically quantified using ImageLab 4.0 (BioRad). Statistical analysis was done using GraphPad Prism 4 to perform one-way ANOVA followed by Dunnett's multiple comparison test. When a higher sensitivity was required, amplicons of expanded pCRISPR arrays were BluePippin (SageScience) size selected and subjected to a second PCR reaction as described previously (Kieper et al. 2018; McKenzie et al.2019).Sequencing of acquired spacersBluePippin extracted and re-amplified expanded CRISPR array amplicons were cloned in the pGemT-easy vector (Promega) and Sanger sequenced (Macrogen Europe, Amsterdam, The Netherlands). Using the Geneious 9.0.5 motif search function, the type I-D repeats were annotated in the sequencing reads and the newly acquired spacers extracted. The origin of newly acquired spacers was determined by nucleotide BLAST search against pCas1–2, pCRISPR and the E. coli BW25113 genome.

Article TitleConserved motifs in the CRISPR leader sequence control spacer acquisition levels in Type I-D CRISPR-Cas systems

Abstract

Integrating short DNA fragments at the correct leader-repeat junction is key to successful CRISPR-Cas memory formation. The Cas1–2 proteins are responsible to carry out this process. However, the CRISPR adaptation process additionally requires a DNA element adjacent to the CRISPR array, called leader, to facilitate efficient localization of the correct integration site. In this work, we introduced the core CRISPR adaptation genescas1andcas2from the Type I-D CRISPR-Cas system ofSynechocystissp. 6803 intoEscherichia coliand assessed spacer integration efficiency. Truncation of the leader resulted in a significant reduction of spacer acquisition levels and revealed the importance of different conserved regions for CRISPR adaptation rates. We found three conserved sequence motifs in the leader of I-D CRISPR arrays that each affected spacer acquisition rates, including an integrase anchoring site. Our findings support the model in which the leader sequence is an integral part of type I-D adaptation inSynechocystissp. acting as a localization signal for the adaptation complex to drive CRISPR adaptation at the first repeat of the CRISPR array.


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