Systematic analysis of Type I-E Escherichia coli CRISPR-Cas PAM sequences ability to promote interference and primed adaptation

Olga Musharova, Vasily Sitnik, Marnix Vlot, Ekaterina Savitskaya, Kirill A. Datsenko, Andrey Krivoy, Ivan Fedorov, Ekaterina Semenova, Stan J.J. Brouns, Konstantin Severinov

Research output: Contribution to journalArticle

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Abstract

CRISPR interference occurs when a protospacer recognized by the CRISPR RNA is destroyed by Cas effectors. In Type I CRISPR-Cas systems, protospacer recognition can lead to «primed adaptation» – acquisition of new spacers from in cis located sequences. Type I CRISPR-Cas systems require the presence of a trinucleotide protospacer adjacent motif (PAM) for efficient interference. Here, we investigated the ability of each of 64 possible trinucleotides located at the PAM position to induce CRISPR interference and primed adaptation by the Escherichia coli Type I-E CRISPR-Cas system. We observed clear separation of PAM variants into three groups: those unable to cause interference, those that support rapid interference and those that lead to reduced interference that occurs over extended periods of time. PAM variants unable to support interference also did not support primed adaptation; those that supported rapid interference led to no or low levels of adaptation, while those that caused attenuated levels of interference consistently led to highest levels of adaptation. The results suggest that primed adaptation is fueled by the products of CRISPR interference. Extended over time interference with targets containing «attenuated» PAM variants provides a continuous source of new spacers leading to high overall level of spacer acquisition.

Original languageEnglish (US)
Pages (from-to)1558-1570
Number of pages13
JournalMolecular microbiology
Volume111
Issue number6
DOIs
StatePublished - Jun 1 2019

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Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas Systems
Escherichia coli
RNA

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology

Cite this

Musharova, Olga ; Sitnik, Vasily ; Vlot, Marnix ; Savitskaya, Ekaterina ; Datsenko, Kirill A. ; Krivoy, Andrey ; Fedorov, Ivan ; Semenova, Ekaterina ; Brouns, Stan J.J. ; Severinov, Konstantin. / Systematic analysis of Type I-E Escherichia coli CRISPR-Cas PAM sequences ability to promote interference and primed adaptation. In: Molecular microbiology. 2019 ; Vol. 111, No. 6. pp. 1558-1570.
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abstract = "CRISPR interference occurs when a protospacer recognized by the CRISPR RNA is destroyed by Cas effectors. In Type I CRISPR-Cas systems, protospacer recognition can lead to «primed adaptation» – acquisition of new spacers from in cis located sequences. Type I CRISPR-Cas systems require the presence of a trinucleotide protospacer adjacent motif (PAM) for efficient interference. Here, we investigated the ability of each of 64 possible trinucleotides located at the PAM position to induce CRISPR interference and primed adaptation by the Escherichia coli Type I-E CRISPR-Cas system. We observed clear separation of PAM variants into three groups: those unable to cause interference, those that support rapid interference and those that lead to reduced interference that occurs over extended periods of time. PAM variants unable to support interference also did not support primed adaptation; those that supported rapid interference led to no or low levels of adaptation, while those that caused attenuated levels of interference consistently led to highest levels of adaptation. The results suggest that primed adaptation is fueled by the products of CRISPR interference. Extended over time interference with targets containing «attenuated» PAM variants provides a continuous source of new spacers leading to high overall level of spacer acquisition.",
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Musharova, O, Sitnik, V, Vlot, M, Savitskaya, E, Datsenko, KA, Krivoy, A, Fedorov, I, Semenova, E, Brouns, SJJ & Severinov, K 2019, 'Systematic analysis of Type I-E Escherichia coli CRISPR-Cas PAM sequences ability to promote interference and primed adaptation', Molecular microbiology, vol. 111, no. 6, pp. 1558-1570. https://doi.org/10.1111/mmi.14237

Systematic analysis of Type I-E Escherichia coli CRISPR-Cas PAM sequences ability to promote interference and primed adaptation. / Musharova, Olga; Sitnik, Vasily; Vlot, Marnix; Savitskaya, Ekaterina; Datsenko, Kirill A.; Krivoy, Andrey; Fedorov, Ivan; Semenova, Ekaterina; Brouns, Stan J.J.; Severinov, Konstantin.

In: Molecular microbiology, Vol. 111, No. 6, 01.06.2019, p. 1558-1570.

Research output: Contribution to journalArticle

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AU - Musharova, Olga

AU - Sitnik, Vasily

AU - Vlot, Marnix

AU - Savitskaya, Ekaterina

AU - Datsenko, Kirill A.

AU - Krivoy, Andrey

AU - Fedorov, Ivan

AU - Semenova, Ekaterina

AU - Brouns, Stan J.J.

AU - Severinov, Konstantin

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N2 - CRISPR interference occurs when a protospacer recognized by the CRISPR RNA is destroyed by Cas effectors. In Type I CRISPR-Cas systems, protospacer recognition can lead to «primed adaptation» – acquisition of new spacers from in cis located sequences. Type I CRISPR-Cas systems require the presence of a trinucleotide protospacer adjacent motif (PAM) for efficient interference. Here, we investigated the ability of each of 64 possible trinucleotides located at the PAM position to induce CRISPR interference and primed adaptation by the Escherichia coli Type I-E CRISPR-Cas system. We observed clear separation of PAM variants into three groups: those unable to cause interference, those that support rapid interference and those that lead to reduced interference that occurs over extended periods of time. PAM variants unable to support interference also did not support primed adaptation; those that supported rapid interference led to no or low levels of adaptation, while those that caused attenuated levels of interference consistently led to highest levels of adaptation. The results suggest that primed adaptation is fueled by the products of CRISPR interference. Extended over time interference with targets containing «attenuated» PAM variants provides a continuous source of new spacers leading to high overall level of spacer acquisition.

AB - CRISPR interference occurs when a protospacer recognized by the CRISPR RNA is destroyed by Cas effectors. In Type I CRISPR-Cas systems, protospacer recognition can lead to «primed adaptation» – acquisition of new spacers from in cis located sequences. Type I CRISPR-Cas systems require the presence of a trinucleotide protospacer adjacent motif (PAM) for efficient interference. Here, we investigated the ability of each of 64 possible trinucleotides located at the PAM position to induce CRISPR interference and primed adaptation by the Escherichia coli Type I-E CRISPR-Cas system. We observed clear separation of PAM variants into three groups: those unable to cause interference, those that support rapid interference and those that lead to reduced interference that occurs over extended periods of time. PAM variants unable to support interference also did not support primed adaptation; those that supported rapid interference led to no or low levels of adaptation, while those that caused attenuated levels of interference consistently led to highest levels of adaptation. The results suggest that primed adaptation is fueled by the products of CRISPR interference. Extended over time interference with targets containing «attenuated» PAM variants provides a continuous source of new spacers leading to high overall level of spacer acquisition.

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