Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli

Yosef, Ido; Goren, Moran G.; Qimron, Udi

doi:10.1093/nar/gks216

Cited by 604 publications

(872 citation statements)

References 27 publications

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“…Csn2 was required for spacer acquisition (Barrangou et al, 2007) and Cas4 is predicted to participate in adaptation (Plagens et al, 2012;Zhang et al, 2012), suggesting that the ability of these strains to acquire new spacers may be compromised. Alternatively, it is possible in theory that this system might still be acquiring new spacers via Cas1-and Cas2-mediated adaptation (Yosef et al, 2012). Indeed, the variable spacer content observed in the type II-C systems between the strains suggests that these systems are still active (Fig.…”

Section: Discussionmentioning

confidence: 99%

Novel configurations of type I and II CRISPR–Cas systems in Corynebacterium diphtheriae

2013

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Clustered regularly interspaced short palindromic repeats (CRISPRs) are major barriers to recombination through recognition of invading nucleic acids, such as phage and plasmids, and promoting their degredation through the action of CRISPR associated (Cas) proteins. The genomic comparison of 17 Corynebacterium diphtheriae strains led to the identification of three novel CRISPR-Cas system variants, based on the Type II (Type II-C) or type I-E systems. The type II-C system was the most common (11/17 isolates) but it lacked the csn2 and cas4 genes that are involved in spacer acquisition. We also identified that this variant type II-C CRISPR-Cas system is present in other bacteria, and the first system was recently characterized in Neisseria meningitidis. In the remaining isolates, the type II-C system was replaced by a variant of type I-E (I-E-a), where the repeat arrays are inserted between the cas3 and cse1 genes. Three isolates with the type II-C system also possess an additional variant of type I-E (I-E-b), elsewhere in the genome, that exhibits a novel divergent gene organization within the cas operon. The nucleotide sequences of the palindromic repeats and the cas1 gene were phylogenetically incongruent to the core genome. The G+C content of the systems is lower (46.0-49.5 mol%) than the overall DNA G+C content (53 mol%), and they are flanked by mobile genetic elements, providing evidence that they were acquired in three independent horizontal gene transfer events. The majority of spacers lack identity with known phage or plasmid sequences, indicating that there is an unexplored reservoir of corynebacteriophages and plasmids. These novel CRISPR-Cas systems may represent a unique mechanism for spacer acquisitions and defence against invading DNA.

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Section: Discussionmentioning

confidence: 99%

Novel configurations of type I and II CRISPR–Cas systems in Corynebacterium diphtheriae

2013

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“…Our analyses targeted large uncharacterized genes proximal to a CRISPR array and cas1, the universal CRISPR integrase [8][9][10] . Among the 155 million protein-coding genes analyzed, we identified the first Cas9 proteins in domain Archaea, and discovered in uncultivated bacteria two new CRISPR-Cas systems, which we refer to as CRISPR-CasX and CRISPR-CasY (Fig.…”

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confidence: 99%

“…C2c3 proteins are putative type V Cas effectors 4 that were identified on short contigs with no taxonomic affiliation, and have not been validated experimentally. Strikingly, both CRISPR-CasY and C2c3 were found next to arrays with short spacers and within loci lacking Cas2, a protein considered essential for integrating DNA into the CRISPR array 9,35 . It remains to be seen whether these type V systems are functional for spacer acquisition.…”

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confidence: 99%

New CRISPR–Cas systems from uncultivated microbes

Burstein

Harrington

Strutt

et al. 2016

Nature

478

369

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CRISPR-Cas systems provide microbes with adaptive immunity by employing short sequences, termed spacers, that guide Cas proteins to cleave foreign DNA 1,2 . Class 2 CRISPR-Cas systems are streamlined versions in which a single Cas protein bound to RNA recognizes and cleaves targeted sequences 3,4 . The programmable nature of these minimal systems has enabled their repurposing as a versatile technology that is broadly revolutionizing biological and clinical research 5 . However, current CRISPR-Cas technologies are based solely on systems from isolated bacteria, leaving untapped the vast majority of enzymes from organisms that have not been cultured. Metagenomics, the sequencing of DNA extracted from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms 6,7 . Here, using genome-resolved metagenomics, we identified novel CRISPR-Cas systems, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in littlestudied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, we discovered two

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“…For each image, we electroporated the pooled oligos into a population of E. coli containing a genomic CRISPR array and expressing the Cas1-Cas2 integrase 18 . Cells were then recovered, passaged overnight, and the next day a sample of the genomic CRISPR arrays were sequenced.…”

mentioning

confidence: 99%

“…3b). Because new spacers are almost always acquired adjacent to the leader sequence in the CRISPR array 18 , pushing previously acquired spacers away from the leader, the order of frames within the GIF can be reconstructed based on the pair-wise order of spacers among many individual arrays.…”

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confidence: 99%

CRISPR–Cas encoding of a digital movie into the genomes of a population of living bacteria

Shipman

Nivala

Macklis

et al. 2017

Nature

276

220

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DNA is an excellent medium for data archival. Recent efforts have illustrated the potential for information storage in DNA using synthesized oligonucleotides assembled in vitro1–6. A relatively unexplored avenue of information storage in DNA is the ability to write information into the genome of a living cell by the addition of nucleotides over time. Using the Cas1-Cas2 integrase, the CRISPR-Cas microbial immune system stores the nucleotide content of invading viruses to confer adaptive immunity7. Harnessed, this system has the potential to write arbitrary information into the genome8. Here, we use the CRISPR-Cas system to encode images and a short movie into the genomes of a population of living bacteria. In doing so, we push the technical limits of this information storage system and optimize strategies to minimize those limitations. We additionally uncover underlying principles of the CRISPR-Cas adaptation system, including sequence determinants of spacer acquisition relevant for understanding both the basic biology of bacterial adaptation as well as its technological applications. This work demonstrates that this system can capture and stably store practical amounts of real data within the genomes of populations of living cells.

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Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli

Cited by 604 publications

References 27 publications

Novel configurations of type I and II CRISPR–Cas systems in Corynebacterium diphtheriae

Novel configurations of type I and II CRISPR–Cas systems in Corynebacterium diphtheriae

New CRISPR–Cas systems from uncultivated microbes

CRISPR–Cas encoding of a digital movie into the genomes of a population of living bacteria

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