Distribution and phasing of sequence motifs that facilitate CRISPR adaptation

Santiago-Frangos, Andrew; Buyukyoruk, Murat; Wiegand, Tanner; Krishna, Pushya; Wiedenheft, Blake

doi:10.1016/j.cub.2021.05.068

Cited by 20 publications

(29 citation statements)

References 79 publications

(121 reference statements)

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“…During CRISPR subtype I-E spacer adaptation, the Cas1-Cas2 complex needs to recognise the boundary between the leading sequence in a CRISPR array and the first repeat (7). This activity is dependent on an Integration Host Factor (IHF) that combines with the leading AT-rich segments and forms a U-shaped structure for recognition (8)(9)(10). In subtype II-A, there is a highly conserved leading-anchor sequence (LAS) located upstream of the first repeat, that ensures all newly integrated spacers are inserted at the leading side (Figure 1B) (11).…”

Section: Introductionmentioning

confidence: 99%

High Frequency of Dynamic Rearrangements In Crispr loci

McInerney

2022

Preprint

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CRISPR-Cas immunization of prokaryotes proceeds by the acquisition of short fragments of invading DNA and integrating them into specific positions within the host genome in a process called adaptation. Adaptation is thought to be polarised, which suggests that CRISPR array spacer order reflects the recentness of the infection. The detailed processes through which CRISPR loci arise, and how they evolve are not completely clear. In this study, we collected 12,461 prokaryotic genomes, and using a combination of four different approaches and a series of conservative filters, we identified CRISPR arrays in 82.7% of Archaea and 40.6% of Bacteria. To understand spacer evolution in these CRISPR loci we firstly tracked point mutations in CRISPR repeats, and secondly, we carried out a comparative analysis of arrays that share multiple similar spacers. Both results indicate that CRISPR arrays are frequently dynamically rearranged. These findings are at odds with a model that suggests that spacer order is likely to reflect the recentness of infection. We conclude that the order of spacers in a CRISPR array, as well as the spacer content of the array, is likely to arise from a combination of events, such as insertion in the middle of the array, recombination within or between arrays, or Horizontal transfer of all or part of an array. We suggest these rearrangements are favoured by natural selection in complex and dynamic environments.

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Section: Introductionmentioning

confidence: 99%

High Frequency of Dynamic Rearrangements In Crispr loci

McInerney

2022

Preprint

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“…It remains unknown if systems that contain additional factors like Cas4 require such host factors. However, various conserved upstream sequence elements have been identified in specific CRISPR-Cas subtypes, including type I-C, and may be important for spacer acquisition as observed in type I-E and I-F (Santiago-Frangos et al, 2021). Further investigation is required to determine whether Cas4-Cas1-Cas2 or a host factor interacts with upstream sequence elements in the CRISPR leader to activate Cas4 and ensure integration fidelity.…”

Section: Discussionmentioning

confidence: 99%

PAM binding ensures orientational integration during Cas4-Cas1-Cas2 mediated CRISPR adaptation

Dhingra

Suresh

Juneja

et al. 2022

Preprint

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Adaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required for selection and processing of DNA segments containing PAM sequences, prior to integration of these "prespacer" substrates as spacers in the CRISPR array. We determined cryo-EM structures of the Cas4-Cas1-Cas2 adaptation complex from the type I-C system that encodes standalone Cas1 and Cas4 proteins. The structures reveal how Cas4 specifically reads out bases within the PAM sequence and how interactions with both Cas1 and Cas2 activate Cas4 endonuclease activity. The Cas4-PAM interaction ensures tight binding between the adaptation complex and the prespacer, significantly enhancing integration of the non-PAM end into the CRISPR array and ensuring correct spacer orientation. Corroborated with our biochemical results, Cas4-Cas1-Cas2 structures with substrates representing various stages of CRISPR adaptation reveal a temporally resolved mechanism for maturation and integration of functional spacers into the CRISPR array.

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“…The only known example was the recruitment of adaptation Cas by IHF in subtype I-E and I-F systems. IHF was found to bind at the leader-proximal region and deform the leader to provide a favourable conformation for the recognition of the Cas1-Cas2 complex [ 18 , 33 ]. It appears that the IHF mediated recruitment of adaptation Cas is an exception because only a few microorganisms encode the ihf gene.…”

Section: Discussionmentioning

confidence: 99%

DNA Motifs and an Accessory CRISPR Factor Determine Cas1 Binding and Integration Activity in Sulfolobus islandicus

Liu

Wang

et al. 2022

IJMS

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CRISPR-Cas systems empower prokaryotes with adaptive immunity against invasive mobile genetic elements. At the first step of CRISPR immunity adaptation, short DNA fragments from the invaders are integrated into CRISPR arrays at the leader-proximal end. To date, the mechanism of recognition of the leader-proximal end remains largely unknown. Here, in the Sulfolobus islandicus subtype I-A system, we show that mutations destroying the proximal region reduce CRISPR adaptation in vivo. We identify that a stem-loop structure is present on the leader-proximal end, and we demonstrate that Cas1 preferentially binds the stem-loop structure in vitro. Moreover, we demonstrate that the integrase activity of Cas1 is modulated by interacting with a CRISPR-associated factor Csa3a. When translocated to the CRISPR array, the Csa3a-Cas1 complex is separated by Csa3a binding to the leader-distal motif and Cas1 binding to the leader-proximal end. Mutation at the leader-distal motif reduces CRISPR adaptation efficiency, further confirming the in vivo function of leader-distal motif. Together, our results suggest a general model for binding of Cas1 protein to a leader motif and modulation of integrase activity by an accessory factor.

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Distribution and phasing of sequence motifs that facilitate CRISPR adaptation

Cited by 20 publications

References 79 publications

High Frequency of Dynamic Rearrangements In Crispr loci

High Frequency of Dynamic Rearrangements In Crispr loci

PAM binding ensures orientational integration during Cas4-Cas1-Cas2 mediated CRISPR adaptation

DNA Motifs and an Accessory CRISPR Factor Determine Cas1 Binding and Integration Activity in Sulfolobus islandicus

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