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

By Rimantas Šapranauskas, G. Gasiunas, C. Fremaux, Rodolphe Barrangou, Philippe Horvath, V. Šikšnys. Nucleic Acids Research, 2011

836 citations recorded by the source corpus.

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.

Subfields: Ecology, Genetics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/d1630766cea694032554717192ab8a840adc66f2

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Citations in the corpus

CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity (2013, background): “This allowed a direct evaluation of the impact of the location and number of mismatches within the 12 bases nearest the PAM region, as well as those in the PAM region (that usually match the canonical NGG motif) (Table 1) on potential off-target activities (8,20).”
CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences (2014, positive): “The lack of activity with PAM-proximal sgRNA bulges in R01 and low levels of activity with PAM-proximal sgRNA bulges in R-30 are consistent with the reduced mismatch tolerance in the ‘seed sequence’ reported in previous studies (9,11,33).”
Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA (2014, background): “Biochemical analyses at the domain levels have enabled site-specific engineering to convert the native Cas9 into a DNA nicking enzyme (Gasiunas et al., 2012; Jinek et al., 2012; Sapranauskas et al., 2011) that facilitates homology-directed repair in eukaryotic cells (Cong et al.”
CRISPR interference (CRISPRi) for sequence-specific control of gene expression (2013, background): “It has been shown that other Cas9 homologs use different PAM sequences 14,24 , and a given CRISPR system may tolerate various PAM sequences during target interference 37,38 .”
Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis (2015, background): “Recently, the Cas9 nuclease (Barrangou et al., 2007; Bolotin et al., 2005; Chylinski et al., 2013, 2014; Deltcheva et al., 2011; Garneau et al., 2010; Gasiunas et al., 2012; Jinek et al., 2012; Sapranauskas et al., 2011) from the microbial type II CRISPR (clustered regularly interspaced short palind”
Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. (2015, neutral): “Several class 2 CRISPR-Cas systems have also been identified and experimentally characterized, but they are all type II and employ homologous RNA-guided endonucleases of the Cas9 family as effectors (Barrangou et al., 2007; Garneau et al., 2010; Deltcheva et al., 2011; Sapranauskas et al., 2011; Jin”
CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity (2014, background): “Also, the ability to readily package and heterologously transfer CRISPR-Cas systems in prokaryotes (Sapranauskas et al., 2011) and even eukaryotes (Cong et al., 2013; Mali et al., 2013b) illustrates the widespread potential of CRISPR-mediated viral resistance, either naturally or by using genetic…”