Structural principles of CRISPR-Cas enzymes used in nucleic acid detection

Das, Anuska; Goswami, Hemant N; Whyms, Charlisa; Sridhara, Sagar; Li, Hong

doi:10.1016/j.jsb.2022.107838

Cited by 14 publications

(4 citation statements)

References 64 publications

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“…6a – c ). Furthermore, unlike several well-characterized Cas12 enzymes that show collateral DNase activities 26 , H750D did not cleave single-stranded DNA (ssDNA) ( Supplementary Fig. 6c ), supporting a tighter control of the RuvC active site in Cas9 than in Cas12.…”

Section: Resultsmentioning

confidence: 74%

“…In addition, several well-characterized Cas12 enzymes, such as Cas12a, show strong ssDNA cutting following activation by cognate dsDNA, an activity that is apparently lacking in Cas9 (ref. 26 ). The only Cas12 enzyme that has been characterized for metal binding so far is Cas12i2, whose RuvC domain both processes guide RNA and cleaves DNA 28 .…”

Section: Discussionmentioning

confidence: 99%

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Coupled catalytic states and the role of metal coordination in Cas9

Das,

Rai,

Roth

et al. 2023

Nat Catal

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Controlling the activity of the CRISPR–Cas9 system is essential to its safe adoption for clinical and research applications. Although the conformational dynamics of Cas9 are known to control its enzymatic activity, details of how Cas9 influences the catalytic processes at both nuclease domains remain elusive. Here we report five cryo-electron microscopy structures of the active Acidothermus cellulolyticus Cas9 complex along the reaction path at 2.2–2.9 Å resolution. We observed that a large movement in one nuclease domain, triggered by the cognate DNA, results in noticeable changes in the active site of the other domain that is required for metal coordination and catalysis. Furthermore, the conformations synchronize the reaction intermediates, enabling coupled cutting of the two DNA strands. Consistent with the roles of conformations in organizing the active sites, adjustments to the metal-coordination residues lead to altered metal specificity of A. cellulolyticus Cas9 and commonly used Streptococcus pyogenes Cas9 in cells.

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Section: Resultsmentioning

confidence: 74%

Section: Discussionmentioning

confidence: 99%

Coupled catalytic states and the role of metal coordination in Cas9

Das,

Rai,

Roth

et al. 2023

Nat Catal

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“…RNase P has been cleverly employed in boosting CRISPR-Cas9 multiplex editing capability by employing it to process transfer RNA-guide RNA (tRNA-gRNA) arrays from a single polycistronic gene toward the generation of numerous gRNAs, thereby contributing to an overall improvement in Cas9's gene editing efficiency (Xie et al, 2015). Such strategies could potentially be explored with other CRISPR-Cas effector complexes such as CRISPR-Cas12, CRISPR-Cas13, and CRISPR-Csm complexes that offer diverse and unique set of functionalities (Chehelgerdi et al, 2024;Das et al, 2022;Makarova et al, 2020;Sridhara et al, 2022;Wang & Yang, 2023;Woodside et al, 2022). The E. coli strain with rnpA knockout served as an efficient means to enhance recombinant protein production of difficult targets including the highly-repetitive glycine-rich spider silk protein, the Cas9 protein and an antibody fragment (Chung et al, 2023).…”

Section: Applications Of Rnase Pmentioning

confidence: 99%

Multiple structural flavors of RNase P in precursor tRNA processing

Sridhara

2024

WIREs RNA

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The precursor transfer RNAs (pre‐tRNAs) require extensive processing to generate mature tRNAs possessing proper fold, structural stability, and functionality required to sustain cellular viability. The road to tRNA maturation follows an ordered process: 5′‐processing, 3′‐processing, modifications at specific sites, if any, and 3′‐CCA addition before aminoacylation and recruitment to the cellular protein synthesis machinery. Ribonuclease P (RNase P) is a universally conserved endonuclease in all domains of life, performing the hydrolysis of pre‐tRNA sequences at the 5′ end by the removal of phosphodiester linkages between nucleotides at position −1 and +1. Except for an archaeal species: Nanoarchaeum equitans where tRNAs are transcribed from leaderless‐position +1, RNase P is indispensable for life and displays fundamental variations in terms of enzyme subunit composition, mechanism of substrate recognition and active site architecture, utilizing in all cases a two metal ion‐mediated conserved catalytic reaction. While the canonical RNA‐based ribonucleoprotein RNase P has been well‐known to occur in bacteria, archaea, and eukaryotes, the occurrence of RNA‐free protein‐only RNase P in eukaryotes and RNA‐free homologs of Aquifex RNase P in prokaryotes has been discovered more recently. This review aims to provide a comprehensive overview of structural diversity displayed by various RNA‐based and RNA‐free RNase P holoenzymes towards harnessing critical RNA–protein and protein–protein interactions in achieving conserved pre‐tRNA processing functionality. Furthermore, alternate roles and functional interchangeability of RNase P are discussed in the context of its employability in several clinical and biotechnological applications.This article is categorized under: RNA Processing > tRNA Processing RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Interactions with Proteins and Other Molecules > RNA‐Protein Complexes

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“…Among the vast kingdom of bacteria, Cas13a (C2c2) found from Leptotrichia shahii (LshCas13a), Leptotrichia buccalis (LbuCas13a) , Lachnospiraceae bacterium (LbaCas13a), Leptotrichia wadei (LwaCas13a), Capnocytophaga canimorsus (CcaCas13b), and Prevotella sp. (PsmCas13b) are currently better understood [39] .…”

Section: Brief Knowledge Of Crispr/cas Systems Based On Cas Proteinsmentioning

confidence: 99%