Expression and purification of A. halodurans Cas1, Cas2 and Cas4

Dhingra, Yukti; Lee, Hayun; Sashital, Dipali G.

doi:10.21769/p1996

Cited by 3 publications

(3 citation statements)

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“…All primers used for cloning various constructs have been listed in Table S1 . Cas4/1 and Cas2 were expressed and purified using previously described procedures for Cas4 and Cas2, respectively ( 47 ).…”

Section: Methodsmentioning

confidence: 99%

Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system

Dhingra,

Sashital

2023

Journal of Biological Chemistry

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Section: Methodsmentioning

confidence: 99%

Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system

Dhingra,

Sashital

2023

Journal of Biological Chemistry

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“…[9] The primary function of Cas4 is to locate and recognize PAM sequences, process captured exogenous DNA fragments to the appropriate length, and remove PAMs before integration, facilitating spacer insertion into the CRISPR array in the correct orientation. [10] As a PAM-specific endonuclease, Cas4 targets both the PAM and non-PAM ends to create an asymmetric complex with Cas1-Cas2, forming an asymmetric substrate. In Pyrococcus furiosus, Cas4-1 acts on the upstream PAM sequence, while Cas4-2 targets the conserved downstream motif, trimming and orienting the double-stranded DNA oligonucleotide pre-spacer region.…”

Section: Cas4 Mediates Part Of the Selection And Processing Of Pam Se...mentioning

confidence: 99%

“…[4] At the PAM end, Cas4 recognizes the PAM sequence within a tight groove through two specific amino acids [12] and modifies the sequence on the PAM side in a Mn 2+ -dependent manner. [10,12] Simultaneously, Cas4's unstable binding at non-PAM ends provides partial protection but delays cleavage upstream of the PAM until integration commences. [4,13] Additionally, in a recent study on Type I-D CRISPR, Cas4 was found to shorten spacer length and aid the CRISPR adaptation complex by supplying DNA substrates tailored for the correct PAM, thereby expanding Cas4's mechanism of action.…”

Section: Cas4 Mediates Part Of the Selection And Processing Of Pam Se...mentioning

confidence: 99%

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

Su,

Jin,

Wang

et al. 2023

Advanced Biology

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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.

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Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system

Dhingra

Sashital

2023

Preprint

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CRISPR-Cas adaptive immune systems uptake short "spacer" sequences from foreign DNA and incorporate them into the host genome to serve as templates for crRNAs that guide interference against future infections. Adaptation in CRISPR systems is mediated by Cas1-Cas2 complexes that catalyze integration of prespacer substrates into the CRISPR array. Many DNA targeting systems also require Cas4 endonucleases for functional spacer acquisition. Cas4 selects prespacers containing a protospacer adjacent motif (PAM) and removes the PAM prior to integration, both of which are required to ensure host immunization. Cas1 has also been shown to function as a nuclease in some systems, but a role for this nuclease activity in adaptation has not been demonstrated. We identified a type I-G Cas4/1 fusion with a nucleolytically active Cas1 domain that can directly participate in prespacer processing. The Cas1 domain is both an integrase and a sequence-independent nuclease that cleaves the non-PAM end of a prespacer, generating optimal overhang lengths that enable integration at the leader side. The Cas4 domain sequence-specifically cleaves the PAM end of the prespacer, ensuring integration of the PAM end at the spacer side. The two domains have varying metal ion requirements. While Cas4 activity is Mn2+ dependent, Cas1 preferentially uses Mg2+ over Mn2+. The dual nuclease activity of Cas4/1 eliminates the need for additional factors in prespacer processing, making the adaptation module self-reliant for prespacer maturation and directional integration.

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Expression and purification of A. halodurans Cas1, Cas2 and Cas4

Cited by 3 publications

References 0 publications

Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system

Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system

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

Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system

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