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Cited by 207CRISPR and Genetic EngineeringBacterial Genetics and BiotechnologyBiochemical and Structural Characterization

The DOI matches its Crossref record; 38 of the 39 references it cites were found in a registry or an index; none of the 47 sources its claims cite is checked for support yet.

The discovery of CRISPR in archaea and bacteria

Open access
Mojica, Francisco J. M., Rodriguez‐Valera, Francisco·2016·The FEBS Journal
DOI 10.1111/febs.13766
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Abstract

CRISPR-Cas are self-/nonself-discriminating systems found in prokaryotic cells. They represent a remarkable example of molecular memory that is hereditarily transmitted. Their discovery can be considered as one of the first fruits of the systematic exploration of prokaryotic genomes. Although this genomic feature was serendipitously discovered in molecular biology studies, it was the availability of multiple complete genomes that shed light about their role as a genetic immune system. Here we tell the story of how this discovery originated and was slowly and painstakingly advanced to the point of understating the biological role of what initially was just an odd genomic feature.

Overview

AI snapshot

Written from the paper’s full text.

TL;DR

This is a historical account by Mojica and Rodríguez-Valera of how CRISPR was discovered in archaea and bacteria, beginning with an unexpected 30-bp repeat pattern in *Haloferax mediterranei* in 1992. It traces how exploratory prokaryotic genomics and spacer-matching analyses led to the recognition of CRISPR-Cas as an adaptive immune system.

Key findings

  • The first CRISPR-like repeats were found serendipitously in *Haloferax mediterranei*: DNA segments 30 bp long repeated at regular distances, with at least 14 almost perfectly conserved repeats flanked at one end by a highly degenerated copy.
  • Similar arrays were already known in bacteria: *E. coli* K12 had two DNA segments with 14 and 7 copies of a 29-bp sequence, and *Mycobacterium bovis* BCG had an array with 49 copies of a 36-bp direct repeat separated by 35–41 bp spacers.
  • By the end of the century, regularly spaced repeats had been discovered in 12 species of archaea and bacteria, with open access to 26 completed microbial genomes; a comprehensive study showed wide distribution and hinted at ancestral origin and high biological relevance.
  • Spacer analysis in *E. coli* showed that about 2% of spacers from 61 additional strains matched sequences in non-CRISPR loci, invariably in genetic elements of the corresponding spacer-carrier species, supporting CRISPR as an immunity system against foreign DNA.

Methods

  • Historical narrative/perspective article, not a primary experimental study; it recounts the authors’ discovery process and the field’s development.
  • Sources include the authors’ recollections, an unpublished Sanger sequencing gel dated 21/08/1992, an unpublished doctoral thesis, and published literature on CRISPR and prokaryotic genomes.
  • Main analysis is chronological and descriptive, linking early repeat observations, genome sequencing advances, spacer database searches, and later experimental demonstrations by other groups.

Limitations

  • Not stated in the paper: the article does not include a formal limitations section.
  • Evident from the text: it is a retrospective personal account, so it may be shaped by recall and perspective rather than systematic review.
  • Evident from the text: some reported experiments are unpublished results, and the authors describe publication rejections and lack of solid experimental evidence at the time, which limits the strength of the early immunity claims.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1111/febs.13766

Cites

38 of 38 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Verification

Claims the paper makes

0 of 47 cited sources checked · 0 supported · 0 flagged

  • Findingnone given

    When experiments equivalent to those in H. volcanii were replicated in E. coli, no clear phenotype was seen.

    “When experiments equivalent to those previously performed in H. volcanii were replicated in E. coli, no clear phenotype was seen.”
    Quote in the paperSubstantial claim
  • Findingown data

    In H. mediterranei, DNA segments 30 bp long were repeated at regular distances.

    “DNA segments, 30 bp long, were repeated at regular distances (Fig. 2A).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [4]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Findingcitation

    In exceptional cases where the cell harboured one of these protospacer-carriers, the similarity between the spacer and its homolog segment was markedly diminished with respect to the protospacer consensus sequence.

    “In the exceptional cases where the cell harboured one of these protospacer-carriers, the similarity between the spacer and its homolog segment was markedly diminished with respect to the protospacer consensus sequence.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [11]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Findingcitation

    The possibility that TREPs serve as frequent recombination sites was dismissed.

    “Firstly, the possibility that TREPs could serve as frequent recombination sites, and hence recombination as the main biological role of TREPs, was dismissed.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [9]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Findingown data

    In H. mediterranei, at least 14 almost perfectly conserved repeats (one occasional mismatch) flanked at one end by a highly degenerated copy were present in the sequenced region.

    “additional sequencing experiments confirmed the presence in this region of at least 14 almost perfectly conserved (one occasional mismatch) repeats flanked at one end by a highly degenerated copy.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [4]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • It may state two separate claims as one. 15
  • Probably not a substantial claim. 8

  • It may state two separate claims as one.

    Findingown data

    Highly variable results were obtained upon deletion of phage-homologous spacers on susceptibility to infection, as well as when analysing transformation efficiency of recombinant plasmids containing spacer-matching sequences.

    “However, highly variable results were obtained upon deletion of phage-homologous spacers on susceptibility to infection, as well as when analysing transformation efficiency of recombinant plasmids containing spacer-matching sequences (unpublished results).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingcitation

    In H. volcanii cultures carrying recombinant plasmids with TREPs, a reduction in cell viability was detected, concomitant with appearance of cells with reduced chromosomal content.

    “Then, a reduction in cell viability detected in these cultures, concomitant with the appearance of cells with reduced chromosomal content, was seen as a sign of incompatibility between replicons containing the repeats, hinting at a role for TREPs in replicon partitioning [9].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [9]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • It may state two separate claims as one.

    Findingcitation

    The spacers of 61 additional E. coli strains, including all available complete genomes, were analyzed, and about 2% of them matched sequences in non-CRISPR loci, invariably in genetic elements of the corresponding spacer-carrier species.

    “To corroborate this finding, the spacers of 61 additional strains, including all available complete genomes, were subjected to the same sort of analysis, turning out that about a 2% of them matched sequences in non-CRISPR loci, invariably in genetic elements of the corresponding spacer-carrier species [11].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [11]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • It may state two separate claims as one.

    Findingnone given

    Repeat binding proteins could not be recovered from cell extracts and topology of recombinant plasmids was not affected by the presence of repeat stretches.

    “However, repeat binding proteins could not be recovered from cell extracts and topology of recombinant plasmids was not affected by the presence of repeat stretches.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingcitation

    A comprehensive study demonstrated the wide distribution of this sort of redundant sequences across a wide microbial diversity, hinting at an ancestral origin and high biological relevance.

    “This comprehensive study demonstrated the wide distribution of this sort of redundant sequences across a wide microbial diversity, hinting at an ancestral origin and high biological relevance [13].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [13]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • It may state two separate claims as one.

    Findingcitation

    TREP sequences were found in the chromosome and a large plasmid of both H. mediterranei and H. volcanii, and similar repeats were present in a second haloarchaeal genus, Haloarcula.

    “TREP sequences were found in the chromosome and a large plasmid of both H. mediterranei and Haloferax volcanii, and hybridization assays disclosed the presence of similar repeats in a second haloarchaeal genus, Haloarcula.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [9]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • It may state two separate claims as one.

    Findingown data

    Northern blots hybridized with probes of the H. mediterranei repeat region revealed multiple transcripts, suggesting highly processed RNAs derived from the repeat arrays.

    “Northern blots hybridized with probes of the repeat region revealed multiple transcripts, suggesting that highly processed RNAs derived from the repeat arrays (Fig. 3).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [4]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisenone given

    Together with Cas proteins, CRISPR are the main constituents of CRISPR-Cas systems that provide prokaryotes with acquired immunity and contribute other unrelated functions such as gene regulation.

    “Together with the CRISPRassociated (Cas) proteins, they are the main constituents of the CRISPR-Cas systems that provide prokaryotes with acquired immunity and contribute other unrelated functions such as gene regulation.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Premisecitation

    These signatures, today known as Protospacer Adjacent Motifs (PAMs), were afterwards shown to be a common feature in diverse CRISPR systems where PAM recognition is a requisite for both spacer uptake and target interference.

    “These signatures, today known as Protospacer Adjacent Motifs (PAMs), were afterwards shown to be a common feature in diverse CRISPR systems [22] where PAM recognition is a requisite for both spacer uptake and target interference [23].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [22]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [23]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Interpretationnone given

    CRISPR was serendipitously discovered in molecular biology studies.

    “Although this genomic feature was serendipitously discovered in molecular biology studies”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Premisecitation

    CRISPR can acquire spacers from, and eventually interfere with, RNA and/or DNA molecules, depending on the particular system.

    “We know now that CRISPR can acquire spacers from, and eventually interfere with, RNA [32] and/or DNA molecules, depending on the particular system.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [32]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim. The paper may give no reason why it holds.

    Interpretationnone given

    CRISPR-Cas represent a remarkable example of molecular memory that is hereditarily transmitted.

    “They represent a remarkable example of molecular memory that is hereditarily transmitted.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Interpretationreasoning

    Taken together, these findings strongly supported a connection between CRISPR and immunity against foreign DNA, most likely guided by CRISPR-RNA molecules.

    “Taken together, these findings strongly supported a connection between CRISPR and immunity against foreign DNA, most likely guided by CRISPR-RNA molecules.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [11]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim. It may be too vague to check against evidence. The paper’s conclusions may not depend on it. The paper may give no reason why it holds.

    Interpretationreasoning

    Their existence proves that prokaryotic genomes are environment tuned.

    “Their existence proves that prokaryotic genomes are environment tuned.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Premisecitation

    Target cleavage is not the only way CRISPR may act; roles beyond immunity, notably through regulation of gene expression, are played by specific systems.

    “Besides, target cleavage is not the only way CRISPR may act and roles beyond immunity, notably through regulation of gene expression [33], are played by specific systems.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [33]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • It may state two separate claims as one.

    Premisecitation

    Regularly spaced repeats had not been seen before in archaea, but similarly arranged structures had been reported in E. coli and Mycobacterium spp.

    “Whereas regularly spaced repeats had not been seen before in archaea, similarly arranged structures had been reported in the chromosome of Gram-negative (Escherichia coli) [5] and Grampositive (Mycobacterium spp.) [6] bacteria.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [5]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [6]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    The average 10% non-coding DNA is present in all these genomes.

    “not to mention the average 10% non-coding DNA [38] present in all these genomes.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [38]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisenone given

    CRISPR form arrays of up to several hundred units in the genome of many bacteria and archaea.

    “Clustered regularly interspaced short palindromic repeats, CRISPR for short, form arrays of up to several hundred units in the genome of many bacteria and archaea.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. The paper may give no reason why it holds.

    Premisenone given

    Molecular cloning was made possible by restriction endonucleases and plasmid vectors, both derived from serendipitous discoveries of prokaryotic cell biology features involved in self/nonself discrimination.

    “It seems appropriate to remember here that the great motor of molecular biology during the XX th century, molecular cloning, was made possible by restriction endonucleases and plasmid vectors, both derived from serendipitous discoveries of prokaryotic cell biology features involved in self/nonself discrimination as well.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper may give no reason why it holds.

    Premisenone given

    CRISPR-Cas are self/non-self discriminating systems found in prokaryotic cells.

    “CRISPR-Cas are self/non-self discriminating systems found in prokaryotic cells.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Interpretationcitation

    Given that protospacers were also detected in chromosomal regions, a regulatory role for CRISPR was inferred, resembling the eukaryotic RNA interference (RNAi) mechanism.

    “Nonetheless, given that protospacers were also detected in chromosomal regions, a regulatory role for CRISPR was inferred, resembling the eukaryotic RNA interference (RNAi) mechanism [11].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [11]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • It may state two separate claims as one.

    Premisecitation

    There are orphan CRISPR arrays in prokaryotic genomes and viruses, as well as CRISPR-like stretches in eukaryotic viruses and mitochondria, most of which are enigmatic regarding their activity or function.

    “There are orphan CRISPR arrays in prokaryotic genomes and viruses [34,35], as well as CRISPR-like stretches in eukaryotic viruses [36] and mitochondria [13], most of which are enigmatic regarding their activity or function.”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [34]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [35]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [36]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [13]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

22 of 22

  • The paper relies on this without support.

    The four selected microorganisms (E. coli, Streptococcus pyogenes, Sulfolobus, and Methanothermobacter thermoautotrophicum) are representative of their respective prokaryotic groups such that common observations about their CRISPR loci apply to prokaryotes in general.

    “Four microorganisms were selected, as representatives of their respective prokaryotic groups, for further analysis: E. coli (Gram-negative bacteria), Streptococcus pyogenes (Gram-positive bacteria), Sulfolobus (crenarchaea) and Methanothermobacter thermoautotrophicum (euryarchaea).”
    “Hence, common observations related to their CRISPR loci would provide conclusions applying to the prokaryotes in general.”
1 of 1

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Self-check results appear here once an independent check has confirmed them.

Cited works that were retracted

Works this paper cites that were later retracted, from the retraction notices publishers deposit. Major when the notice came out before this paper was published.

  • Cites a retracted work: 10.1038/nbt.3547

    Info

    The cited work was retracted on 2017-07-26 (notice 10.1038/nbt0817-797a), not before this paper was published (2016-06-15).

    Cited work 10.1038/nbt.3547 · retracted 2017-07-26 · notice 10.1038/nbt0817-797a · this paper published 2016-06-15

1 of 1

Citations

Cites

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Flagged first, then by citations

1 of the 39 references couldn’t be matched to a known paper.

Cited by

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Related

Metadata

Authors
Mojica, F. J. M., & Rodriguez‐Valera, F.
Affiliations
  • University of Alicante (ES)
  • Evolutionary Genomics Group Universidad Miguel Hernandez Alicante Spain
Corresponding author
Mojica, Francisco J. M.
ORCID
Mojica, Francisco J. M.
Venue
The FEBS Journal
Publisher
Wiley
Published
2016-06-15
Volume
283
Issue
17
Pages
3162-3169
ISSN
  • 1742-464X (ISSN-L)
  • 1742-4658
DOI
10.1111/febs.13766
OpenAlex
W2397372517
Cited by
207
References
39
Citations by year
2026: 10 · 2025: 24 · 2024: 27 · 2023: 22 · 2022: 36 · 2021: 26 · 2020: 19 · 2019: 11 · 2018: 18 · 2017: 11 · 2016: 3
FWCI
1.24
Type
journal article
Language
en
Open access
Licence
Locations
  • FEBS Journal (publishedVersion, other-oa)
  • PubMed (publishedVersion)
  • LA Referencia (Red Federada de Repositorios Institucionales de Publicaciones Científicas) (submittedVersion)
  • Repositorio Institucional de la Universidad de Alicante (Universidad de Alicante) (submittedVersion)
Funders
  • European Commission (291815)
  • Seventh Framework Programme
  • Generalitat Valenciana
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
Keywords
CRISPR-Cas, archaea, bacteria, self/non-self discrimination, molecular memory, adaptive immunity
MeSH terms
Archaea, Bacteria, Genome, Bacterial, Genome, Archaeal, Genomics, CRISPR-Cas Systems

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