The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli

Abstract

The CRISPR/Cas adaptive immune system provides resistance against phages and plasmids in Archaea and Bacteria. CRISPR loci integrate short DNA sequences from invading genetic elements that provide small RNA-mediated interference in subsequent exposure to matching nucleic acids. In Streptococcus thermophilus, it was previously shown that the CRISPR1/Cas system can provide adaptive immunity against phages and plasmids by integrating novel spacers following exposure to these foreign genetic elements that subsequently direct the specific cleavage of invasive homologous DNA sequences. Here, we show that the S. thermophilus CRISPR3/Cas system can be transferred into Escherichia coli and provide heterologous protection against plasmid transformation and phage infection. We show that interference is sequence-specific, and that mutations in the vicinity or within the proto-spacer adjacent motif (PAM) allow plasmids to escape CRISPR-encoded immunity. We also establish that cas9 is the sole cas gene necessary for CRISPR-encoded interference. Furthermore, mutation analysis revealed that interference relies on the Cas9 McrA/HNH- and RuvC/RNaseH-motifs. Altogether, our results show that active CRISPR/Cas systems can be transferred across distant genera and provide heterologous interference against invasive nucleic acids. This can be leveraged to develop strains more robust against phage attack, and safer organisms less likely to uptake and disseminate plasmid-encoded undesirable genetic elements.

Figure 1.

CRISPR/Cas systems of S. thermophilus DGCC7710. Cas proteins of the CRISPR1 and CRISPR3 systems belong to Type II, while CRISPR2 and CRISPR4 belong to Type III and Type I, respectively.

Figure 2.

CRISPR3/Cas system of S. thermophilus provides immunity against plasmid transformation in E. coli cells. (A) Schematic representation of CRISPR3/Cas system cloning and construction of the plasmids for interference assay. Streptococcus thermophilus CRISPR3/Cas system was cloned into E. coli plasmid pACYC184. Plasmids for interference assays were obtained by inserting a proto-spacer and PAM into pUC18 plasmid. (B) Schematic representation of the plasmid transformation interference assay. Escherichia coli RR1 recipient strains carrying plasmids pCRISPR3 and pACYC184 with and without the S. thermophilus CRISPR3/Cas system, respectively, were transformed with plasmids pSP1 and pSP2 carrying proto-spacers and PAMs or pUC18. (C) Interference of plasmid transformation by S. thermophilus CRISPR3/Cas system in E. coli cells. Transformation efficiency is expressed as cfu per nanogram of plasmid DNA (mean ± SD).

Figure 3.

Impact of proto-spacer and PAM mutations on CRISPR-encoded plasmid immunity. (A) Effect of mutations in the proto-spacer region on the plasmid transformation efficiency. Mutations are shown schematically above the figure. (B) Effect of mutations in the PAM region on the plasmid transformation efficiency.

Figure 4.

Mutational analysis of cas genes. (A) Schematic representation of plasmids carrying mutant variants of cas genes. Individual genes were disrupted by frameshift mutations or small deletions. Three cas genes (cas1, cas2, csn2) were removed by deletion. (B) cas9 gene alone prevents plasmid DNA transformation. (C) Mutagenesis of Cas9 protein. Conserved domains and mutated amino acids are indicated. (D) Mutations in the conserved RuvC/RNaseH− and McrA/HNH domains inactivate cas9.