Mechanism for Cas4-assisted directional spacer acquisition in CRISPR–Cas

Hu, Chunyi; Almendros, Cristóbal; Nam, Ki Hyun; Costa, Ana Rita; Vink, Jochem N.A.; Haagsma, Anna C.; Bagde, Saket R.; Brouns, Stan J. J.; Ke, Ailong

doi:10.1038/s41586-021-03951-z

Cited by 42 publications

(89 citation statements)

References 58 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We initially reconstituted a complex bound to a prespacer substrate comprising a 22-base pair (bp) duplex with unprocessed 15 nucleotide (nt) 3′ overhangs, each containing the 5′-GAA-3′ PAM sequence beginning at the seventh position of the overhangs ( Figures 1E and S1A-C ). Consistent with our previous negative stain reconstruction and the recent cryo-EM structure of the type I-G Cas4/1-Cas2 complex (Hu et al, 2021; Lee et al, 2019), the cryo-EM structure reveals the typical butterfly shape of Cas1-Cas2. Two Cas1 dimers flank a Cas2 dimer in the middle and a single Cas4 subunit is associated with the wing tip on one end of each Cas1 dimer, resulting in a stoichiometry of Cas4 2 :Cas1 4 :Cas2 2 ( Figures 1D and 1E ).…”

Section: Resultssupporting

confidence: 88%

“…Cryo-EM density for the Cas4 subunits was sufficiently high resolution to build a structural model for a type I-C Cas4 protein using an AlphaFold predicted structural model for guidance ( Figure S3A ) (Jumper et al, 2021; Mirdita et al, 2022; Varadi et al, 2022). Type I-C Cas4 adopts an overall fold similar to previously determined structures of type I-A, I-B and I-G Cas4 proteins (Hu et al, 2021; Lemak et al, 2013, 2014). The C-terminus of type I-C Cas4 extends further than in the other structures, adopting an α-helix ( Figures 1E and S3B-C ).…”

Section: Resultssupporting

confidence: 74%

“…Target binding and degradation of the invading nucleic acids is preceded by recognition of the PAM (protospacer adjacent motif) located adjacent to the target sequence (Mojica et al, 2009; Redding et al, 2015; Sashital et al, 2012; Semenova et al, 2011; Sternberg et al, 2014; Xue et al, 2017). Because PAM recognition is required for interference, spacers acquired during adaptation must be selected from “prespacers” that contain a PAM sequence (Hu et al, 2021; Wang et al, 2015; Xiao et al, 2017). Importantly, the PAM is not incorporated into the CRISPR array, preventing “self”-targeting by the interference machinery and necessitating precise removal of the PAM before integration.…”

Section: Introductionmentioning

confidence: 99%

“…Adaptation is mediated by the Cas1-Cas2 integration complex. Cas1 and Cas2 proteins form a heterohexameric complex with two distal Cas1 dimers flanking a Cas2 dimer (Fagerlund et al, 2017; Hu et al, 2021; Lee et al, 2019; Nuñez et al, 2014; Rollins et al, 2017; Xiao et al, 2017). While cas1 and cas2 are nearly universally conserved in diverse CRISPR-Cas systems, additional factors are required for adaptation in various sub-types (Makarova et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

“…Cas4 forms a higher order complex with Cas1-Cas2 in the presence of a dsDNA substrate, and is only activated as an endonuclease within this complex (Lee et al, 2019). Recent cryo-electron microscopy (cryo-EM) structures of an adaptation complex from the type I-G system demonstrated that Cas4/1 fusion proteins enable polarized integration through activation of Cas4 cleavage following the first integration event into the CRISPR array (Hu et al, 2021). However, it remains unclear how systems with standalone Cas4 and Cas1 proteins activate Cas4 as an endonuclease for PAM cleavage and enable polarized integration of new spacers.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

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

Dhingra

Suresh

Juneja

et al. 2022

Preprint

View full text Add to dashboard Cite

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.

show abstract

Section: Resultssupporting

confidence: 88%

Section: Resultssupporting

confidence: 74%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Dhingra

Suresh

Juneja

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

Unraveling Exogenous DNA Processing in Cas4‐Lacking Crispr Systems: A Novel Bypass Pathway Explored

Su,

Jin,

Wang

et al. 2023

Advanced Biology

View full text Add to dashboard Cite

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated (Cas) systems are widely distributed adaptive immune systems found in prokaryotes. The process involves three main stages: adaptation, expression, and interference. While the adaptation stage has been extensively studied, there is still an incomplete understanding of the mechanisms underlying the capture, trimming, and integration of exogenous DNA. For instance, Cas4, a CRISPR‐Cas protein with endonuclease activity, is responsible for selecting and processing protospacer adjacent motif (PAM) sequences. However, some CRISPR isoforms lack Cas4 activity, relying on other enzymes for adaptive immunity. Recently, Wang et al. presented a novel model of exogenous DNA processing in a type I‐E CRISPR system lacking Cas4 in a Nature article. This model integrates protospacer processing into CRISPR arrays through fine‐tuned synthases formed by DnaQ‐like exonuclease (DEDDh) and Cas1‐Cas2 complexes. Their study introduces a novel model, shedding new light on the evolution of CRISPR adaptive immunity. This perspective comprehensively examines the fundamental process of CRISPR adaptive immunity, detailing both the classical pathway mediated by Cas4 and the alternative pathway mediated by DEDDh. Furthermore, a thorough evaluation of Wang et al.’s work is conducted, highlighting its strengths, weaknesses, and existing research challenges.

show abstract