scholarly journals Cas3/I-C mediated target DNA recognition and cleavage during CRISPR interference are independent of the composition and architecture of Cascade surveillance complex

2020 ◽  
Vol 48 (5) ◽  
pp. 2486-2501 ◽  
Author(s):  
Siddharth Nimkar ◽  
B Anand
Keyword(s):  
Nuclease Activity ◽  
Helicase Domain ◽  
Type I ◽  
Conserved Residues ◽  
Crispr Interference ◽  
Target Strand ◽  
Target Dna ◽  
Evolutionarily Conserved ◽  
Single Stranded Region

Abstract In type I CRISPR-Cas system, Cas3—a nuclease cum helicase—in cooperation with Cascade surveillance complex cleaves the target DNA. Unlike the Cascade/I-E, which is composed of five subunits, the Cascade/I-C is made of only three subunits lacking the CRISPR RNA processing enzyme Cas6, whose role is assumed by Cas5. How these differences in the composition and organization of Cascade subunits in type I-C influence the Cas3/I-C binding and its target cleavage mechanism is poorly understood. Here, we show that Cas3/I-C is intrinsically a single-strand specific promiscuous nuclease. Apart from the helicase domain, a constellation of highly conserved residues—which are unique to type I-C—located in the uncharacterized C-terminal domain appears to influence the nuclease activity. Recruited by Cascade/I-C, the HD nuclease of Cas3/I-C nicks the single-stranded region of the non-target strand and positions the helicase motor. Powered by ATP, the helicase motor reels in the target DNA, until it encounters the roadblock en route, which stimulates the HD nuclease. Remarkably, we show that Cas3/I-C supplants Cas3/I-E for CRISPR interference in type I-E in vivo, suggesting that the target cleavage mechanism is evolutionarily conserved between type I-C and type I-E despite the architectural difference exhibited by Cascade/I-C and Cascade/I-E.

scholarly journals Cas3 Mediated Target DNA Recognition and Cleavage is Independent of the Composition and Architecture of Cascade Surveillance Complex

2019 ◽  
Author(s):  
Siddharth Nimkar ◽  
B. Anand
Keyword(s):  
Rna Processing ◽  
Dna Recognition ◽  
Nuclease Activity ◽  
Helicase Domain ◽  
Type I ◽  
Conserved Residues ◽  
Target Dna ◽  
Evolutionarily Conserved ◽  
Single Stranded Region

ABSTRACTIn type I CRISPR-Cas system, Cas3 –a nuclease cum helicase– in cooperation with Cascade surveillance complex cleaves the target DNA. Unlike the Cascade/I-E, which is composed of five subunits, the Cascade/I-C is made of only three subunits lacking the CRISPR RNA processing enzyme Cas6, whose role is assumed by Cas5. How these differences in the composition and organisation of Cascade subunits in type I-C influences the Cas3/I-C binding and its target cleavage mechanism is poorly understood. Here, we show that Cas3/I-C is intrinsically a single-strand specific promiscuous nuclease. Apart from the helicase domain, a constellation of highly conserved residues –that are unique to type I-C– located in the uncharacterised C-terminal domain appears to influence the nuclease activity. Recruited by Cascade/I-C, the HD nuclease of Cas3/I-C nicks the single-stranded region of the nontarget strand and positions the helicase motor. Powered by ATP, the helicase motor reels in the target DNA, until it encounters the roadblock en route, which stimulates the HD nuclease. Remarkably, we show that Cas3/I-C supplants Cas3/I-E for CRISPR interference in type I-E in vivo, suggesting that the target cleavage mechanism is evolutionarily conserved between type I-C and type I-E despite the architectural difference exhibited by Cascade/I-C and Cascade/I-E.

scholarly journals Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3

2021 ◽  
Author(s):  
Kazuto Yoshimi ◽  
Kohei TAKESHITA ◽  
Noriyuki Kodera ◽  
Satomi Shibumura ◽  
Yuko Yamauchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Speed ◽  
Dna Helicase ◽  
Type I ◽  
Loop Formation ◽  
Force Microscopy ◽  
Atomic Force ◽  
Target Strand ◽  
Target Dna

Type I CRISPR-Cas3 uses an RNA-guided multi Cas-protein complex, Cascade, which detects and degrades foreign nucleic acids via the helicase-nuclease Cas3 protein. Despite many studies using cryoEM and smFRET, the precise mechanism of Cas3-mediated cleavage and degradation of target DNA remains elusive. Here we reconstitute the CRISPR-Cas3 system in vitro to show how the Escherichia coli Cas3 (EcoCas3) with EcoCascade exhibits collateral non-specific ssDNA cleavage and target specific DNA degradation. Partial binding of EcoCascade to target DNA with tolerated mismatches within the spacer sequence, but not the PAM, elicits collateral ssDNA cleavage activity of recruited EcoCas3. Conversely, stable binding with complete R-loop formation drives EcoCas3 to nick the non-target strand (NTS) in the bound DNA. Helicase-dependent unwinding then combines with trans ssDNA cleavage of the target strand and repetitive cis cleavage of the NTS to degrade the target dsDNA substrate. High-speed atomic force microscopy demonstrates that EcoCas3 bound to EcoCascade repeatedly reels and releases the target DNA, followed by target fragmentation. Together, these results provide a revised model for collateral ssDNA cleavage and target dsDNA degradation by CRISPR-Cas3, furthering understanding of type I CRISPR priming and interference and informing future genome editing tools.

scholarly journals The role of Cas8 in type I CRISPR interference

2015 ◽  
Vol 35 (3) ◽  
Author(s):  
Simon D.B. Cass ◽  
Karina A. Haas ◽  
Britta Stoll ◽  
Omer S. Alkhnbashi ◽  
Kundan Sharma ◽  
...  
Keyword(s):  
Nuclease Activity ◽  
Type I ◽  
Crispr Interference ◽  
Archaeal Species ◽  
Nucleoprotein Complex ◽  
Biochemical Analyses

We have used genetic and protein biochemical analyses in two archaeal species to identify that Cas8 is essential for CRISPR interference. We provide evidence that Cas8 functions as part of archaeal Cascade, an R-loop forming nucleoprotein complex, recognizing protospacer adjacent motifs (PAMs) on invader DNA. An RNA nuclease activity of Cas8 is also described.

scholarly journals Genome editing in plants using CRISPR type I-D nuclease

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Keishi Osakabe ◽  
Naoki Wada ◽  
Tomoko Miyaji ◽  
Emi Murakami ◽  
Kazuya Marui ◽  
...  
Keyword(s):  
Genome Editing ◽  
First Generation ◽  
Genome Engineering ◽  
Targeted Mutagenesis ◽  
Plant Genome ◽  
Type I ◽  
Crispr System ◽  
Target Dna ◽  
Short Indels

Abstract Genome editing in plants has advanced greatly by applying the clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas system, especially CRISPR-Cas9. However, CRISPR type I—the most abundant CRISPR system in bacteria—has not been exploited for plant genome modification. In type I CRISPR-Cas systems, e.g., type I-E, Cas3 nucleases degrade the target DNA in mammals. Here, we present a type I-D (TiD) CRISPR-Cas genome editing system in plants. TiD lacks the Cas3 nuclease domain; instead, Cas10d is the functional nuclease in vivo. TiD was active in targeted mutagenesis of tomato genomic DNA. The mutations generated by TiD differed from those of CRISPR/Cas9; both bi-directional long-range deletions and short indels mutations were detected in tomato cells. Furthermore, TiD can be used to efficiently generate bi-allelic mutant plants in the first generation. These findings indicate that TiD is a unique CRISPR system that can be used for genome engineering in plants.

scholarly journals Highly efficient primed spacer acquisition from targets destroyed by the Escherichia coli type I-E CRISPR-Cas interfering complex

2016 ◽  
Vol 113 (27) ◽  
pp. 7626-7631 ◽  
Author(s):  
Ekaterina Semenova ◽  
Ekaterina Savitskaya ◽  
Olga Musharova ◽  
Alexandra Strotskaya ◽  
Daria Vorontsova ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Functional Outcomes ◽  
Effector Proteins ◽  
Type I ◽  
Crispr Interference ◽  
Highly Efficient ◽  
Target Dna ◽  
Short Palindromic Repeat ◽  
Rapid Destruction ◽  
Foreign Dna

Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated (Cas) immunity relies on adaptive acquisition of spacers—short fragments of foreign DNA. For the type I-E CRISPR-Cas system from Escherichia coli, efficient “primed” adaptation requires Cas effector proteins and a CRISPR RNA (crRNA) whose spacer partially matches a segment (protospacer) in target DNA. Primed adaptation leads to selective acquisition of additional spacers from DNA molecules recognized by the effector–crRNA complex. When the crRNA spacer fully matches a protospacer, CRISPR interference—that is, target destruction without acquisition of additional spacers—is observed. We show here that when the rate of degradation of DNA with fully and partially matching crRNA targets is made equal, fully matching protospacers stimulate primed adaptation much more efficiently than partially matching ones. The result indicates that different functional outcomes of CRISPR-Cas response to two kinds of protospacers are not caused by different structures formed by the effector–crRNA complex but are due to the more rapid destruction of targets with fully matching protospacers.

scholarly journals Direct visualization of native CRISPR target search in live bacteria reveals Cascade DNA surveillance mechanism

2019 ◽  
Author(s):  
Jochem N.A. Vink ◽  
Koen J.A. Martens ◽  
Marnix Vlot ◽  
Rebecca E. McKenzie ◽  
Cristóbal Almendros ◽  
...  
Keyword(s):  
Search Time ◽  
Type I ◽  
Exponential Relationship ◽  
Target Search ◽  
Rna Surveillance ◽  
Crispr Interference ◽  
Dna Elements ◽  
Cellular Dna ◽  
Surveillance Mechanism

AbstractCRISPR-Cas systems encode RNA-guided surveillance complexes to find and cleave invading DNA elements. While it is thought that invaders are neutralized minutes after cell entry, the mechanism and kinetics of target search and its impact on CRISPR protection levels have remained unknown. Here we visualized individual Cascade complexes in a native type I CRISPR-Cas system. We uncovered an exponential relationship between Cascade copy number and CRISPR interference levels, pointing to a time-driven arms race between invader replication and target search, in which 20 Cascade complexes provide 50% protection. Driven by PAM-interacting subunit Cas8e, Cascade spends half its search time rapidly probing DNA (∼30 ms) in the nucleoid. We further demonstrate that target DNA transcription and CRISPR arrays affect the integrity of Cascade and impact CRISPR interference. Our work establishes the mechanism of cellular DNA surveillance by Cascade that allows the timely detection of invading DNA in a crowded, DNA-packed environment.One sentence summaryThe results from in vivo tracking of single CRISPR RNA-surveillance complexes in the native host cell explain their ability to rapidly recognize invader sequences.

scholarly journals Determining the Specificity of Cascade Binding, Interference, and Primed Adaptation In Vivo in the Escherichia coli Type I-E CRISPR-Cas System

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
pp. e02100-17 ◽  
Author(s):  
Lauren A. Cooper ◽  
Anne M. Stringer ◽  
Joseph T. Wade
Keyword(s):  
Escherichia Coli ◽  
Protein Complex ◽  
Dna Interaction ◽  
Type I ◽  
Base Pairing ◽  
Content Type ◽  
E Coli ◽  
Target Dna ◽  
Target Sites

ABSTRACT In clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) immunity systems, short CRISPR RNAs (crRNAs) are bound by Cas proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptation in vivo, for the type I-E system of Escherichia coli. Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target. Consequently, a single crRNA promotes Cascade association with numerous off-target sites, and the endogenous E. coli crRNAs direct Cascade binding to >100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, >18 bp are required for both interference and primed adaptation. Hence, Cascade binding to suboptimal, off-target sites is inert. Our data support a model in which the initial Cascade association with DNA targets requires only limited sequence complementarity at the crRNA 5′ end whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and primed adaptation, requires extensive base pairing. IMPORTANCE Many bacterial and archaeal species encode CRISPR-Cas immunity systems that protect against invasion by foreign DNA. In the Escherichia coli CRISPR-Cas system, a protein complex, Cascade, binds 61-nucleotide (nt) CRISPR RNAs (crRNAs). The Cascade complex is directed to invading DNA molecules through base pairing between the crRNA and target DNA. This leads to recruitment of the Cas3 nuclease, which destroys the invading DNA molecule and promotes acquisition of new immunity elements. We made the first in vivo measurements of Cascade binding to DNA targets. Thus, we show that Cascade binding to DNA is highly promiscuous; endogenous E. coli crRNAs can direct Cascade binding to >100 chromosomal locations. In contrast, we show that targeted degradation and acquisition of new immunity elements require highly specific association of Cascade with DNA, limiting CRISPR-Cas function to the appropriate targets.

scholarly journals An RNA pseudoknot is essential for standby-mediated translation of the tisB toxin mRNA in Escherichia coli

2020 ◽  
Vol 48 (21) ◽  
pp. 12336-12347
Author(s):  
Cédric Romilly ◽  
Anne Lippegaus ◽  
E Gerhart H Wagner
Keyword(s):  
Escherichia Coli ◽  
Point Mutations ◽  
Essential Elements ◽  
Toxin Gene ◽  
Type I ◽  
Escherichia Coli Cells ◽  
Mrna Variants ◽  
Single Stranded Region ◽  
Rna Pseudoknot

Abstract In response to DNA damage, Escherichia coli cells activate the expression of the toxin gene tisB of the toxin–antitoxin system tisB-istR1. Of three isoforms, only the processed, highly structured +42 tisB mRNA is active. Translation requires a standby site, composed of two essential elements: a single-stranded region located 100 nucleotides upstream of the sequestered RBS, and a structure near the 5′-end of the active mRNA. Here, we propose that this 5′-structure is an RNA pseudoknot which is required for 30S and protein S1-alone binding to the mRNA. Point mutations that prevent formation of this pseudoknot inhibit formation of translation initiation complexes, impair S1 and 30S binding to the mRNA, and render the tisB mRNA non-toxic in vivo. A set of mutations created in either the left or right arm of stem 2 of the pseudoknot entailed loss of toxicity upon overexpression of the corresponding mRNA variants. Combining the matching right-left arm mutations entirely restored toxicity levels to that of the wild-type, active mRNA. Finally, since many pseudoknots have high affinity for S1, we predicted similar pseudoknots in non-homologous type I toxin–antitoxin systems that exhibit features similar to that of tisB-IstR1, suggesting a shared requirement for standby acting at great distances.

scholarly journals FIP200 restricts RNA virus infection by facilitating RIG-I activation

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Lingyan Wang ◽  
Kun Song ◽  
Wenzhuo Hao ◽  
Yakun Wu ◽  
Girish Patil ◽  
...  
Keyword(s):  
Virus Infection ◽  
Rna Virus ◽  
Innate Immune ◽  
Host Susceptibility ◽  
In Vivo Studies ◽  
Helicase Domain ◽  
Type I ◽  
Immune Signaling ◽  
Innate Immune Signaling

AbstractRetinoic acid-inducible gene I (RIG-I) senses viral RNA and instigates an innate immune signaling cascade to induce type I interferon expression. Currently, the regulatory mechanisms controlling RIG-I activation remain to be fully elucidated. Here we show that the FAK family kinase-interacting protein of 200 kDa (FIP200) facilitates RIG-I activation. FIP200 deficiency impaired RIG-I signaling and increased host susceptibility to RNA virus infection. In vivo studies further demonstrated FIP200 knockout mice were more susceptible to RNA virus infection due to the reduced innate immune response. Mechanistic studies revealed that FIP200 competed with the helicase domain of RIG-I for interaction with the two tandem caspase activation and recruitment domains (2CARD), thereby facilitating the release of 2CARD from the suppression status. Furthermore, FIP200 formed a dimer and facilitated 2CARD oligomerization, thereby promoting RIG-I activation. Taken together, our study defines FIP200 as an innate immune signaling molecule that positively regulates RIG-I activation.

scholarly journals Determining the specificity of Cascade binding, interference, and primed adaptation in vivo in the Escherichia coli type I-E CRISPR-Cas system

2017 ◽  
Author(s):  
Lauren A. Cooper ◽  
Anne M. Stringer ◽  
Joseph T. Wade
Keyword(s):  
Escherichia Coli ◽  
Protein Complex ◽  
Dna Interaction ◽  
Type I ◽  
Base Pairing ◽  
E Coli ◽  
Target Dna ◽  
Target Sites ◽  
Sequence Complementarity

ABSTRACTIn CRISPR-Cas immunity systems, short CRISPR RNAs (crRNAs) are bound by CRISPR-associated (Cas) proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptation in vivo, for the type I-E system of Escherichia coli. Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target. Consequently, a single crRNA promotes Cascade association with numerous off-target sites, and the endogenous E. coli crRNAs direct Cascade binding to >100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, >18 bp are required for both interference and primed adaptation. Hence, Cascade binding to sub-optimal, off-target sites is inert. Our data support a model in which initial Cascade association with DNA targets requires only limited sequence complementarity at the crRNA 5□ end, whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and primed adaptation, requires extensive base-pairing.IMPORTANCEMany bacterial and archaeal species encode CRISPR-Cas immunity systems that protect against invasion by foreign DNA. In the Escherichia coli CRISPR-Cas system, a protein complex, Cascade, binds 61 nt CRISPR RNAs (crRNAs). The Cascade complex is directed to invading DNA molecules through base-pairing between the crRNA and target DNA. This leads to recruitment of the Cas3 nuclease that destroys the invading DNA molecule and promotes acquisition of new immunity elements. We make the first in vivo measurements of Cascade binding to DNA targets. Thus, we show that Cascade binding to DNA is highly promiscuous; endogenous E. coli crRNAs can direct Cascade binding to >100 chromosomal locations. By contrast, we show that target degradation and acquisition of new immunity elements requires highly specific association of Cascade with DNA, limiting CRISPR-Cas function to the appropriate targets.

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