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

Dhingra, Yukti; Sashital, Dipali G.

doi:10.1016/j.jbc.2023.105178

Cited by 2 publications

References 48 publications

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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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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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Branched DNA Processing by a Thermostable CAS-Cas4 fromThermococcus onnurineus: Expanding Biochemical Landscape of Nuclease Activity

Jain,

Pattnayak,

Aggarwal

et al. 2024

Preprint

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The adaptive immune function of CRISPR-Cas systems in bacteria and archaea is mediated through CRISPR-Associated Proteins (Cas). The adaptation module, typically involving Cas1, Cas2, and Cas4, helps integrate viral spacer sequences into the host genome. Cas4 proteins are classified into two types based on neighboring genes: CAS-Cas4, flanked by other cas genes, and Solo-Cas4, which exist independently. While CAS-Cas4 proteins are implicated in adaptation, they remain biochemically uncharacterized in archaea, unlike archaeal Solo-Cas4 proteins. This study biochemically characterizes TON_0321, a CAS-Cas4 protein from the Type IV-C CRISPR cassette of Thermococcus onnurineus. TON_0321 exhibits 5′ to 3′ exonuclease activity and unique structure-dependent endonuclease activity, shedding light on CAS-Cas4 functional diversity. A distinct spatial organization of the catalytic site, angled with the positively charged patch on the protein surface, enables TON_0321 to recognize branching points in DNA substrates. Furthermore, this spatial arrangement facilitates cleavage 2 to 3 nucleotides away from the branch point in the 5′ direction, demonstrating structure-specific endonuclease activity.

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

Cited by 2 publications

References 48 publications

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

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

Branched DNA Processing by a Thermostable CAS-Cas4 fromThermococcus onnurineus: Expanding Biochemical Landscape of Nuclease Activity

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