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EcoSal Plus

Domain 7:

Genetics and Genetic Tools

Mechanisms of Type I-E and I-F CRISPR-Cas Systems in

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.
  • Authors: Chaoyou Xue1,2, and Dipali G. Sashital3
  • Editors: James M. Slauch4, Gregory Phillips5
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Roy J. Carver Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, IA; 2: Present address: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY; 3: Roy J. Carver Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, IA; 4: The School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, Urbana, IL; 5: College of Veterinary Medicine, Iowa State University, Ames, IA
  • Received 25 June 2018 Accepted 02 January 2019 Published 06 February 2019
  • Address correspondence to Dipali Sashital, [email protected]
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  • Abstract:

    CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against invasion by bacteriophages and other mobile genetic elements. Short fragments of invader DNA are stored as immunological memories within CRISPR (clustered regularly interspaced short palindromic repeat) arrays in the host chromosome. These arrays provide a template for RNA molecules that can guide CRISPR-associated (Cas) proteins to specifically neutralize viruses upon subsequent infection. Over the past 10 years, our understanding of CRISPR-Cas systems has benefited greatly from a number of model organisms. In particular, the study of several members of the Gram-negative family, especially and , have provided significant insights into the mechanisms of CRISPR-Cas immunity. In this review, we provide an overview of CRISPR-Cas systems present in members of the . We also detail the current mechanistic understanding of the type I-E and type I-F CRISPR-Cas systems that are commonly found in enterobacteria. Finally, we discuss how phages can escape or inactivate CRISPR-Cas systems and the measures bacteria can enact to counter these types of events.

  • Citation: Xue C, Sashital D. 2019. Mechanisms of Type I-E and I-F CRISPR-Cas Systems in , EcoSal Plus 2019; doi:10.1128/ecosalplus.ESP-0008-2018

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/content/journal/ecosalplus/10.1128/ecosalplus.ESP-0008-2018
2019-02-06
2021-05-14

Abstract:

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against invasion by bacteriophages and other mobile genetic elements. Short fragments of invader DNA are stored as immunological memories within CRISPR (clustered regularly interspaced short palindromic repeat) arrays in the host chromosome. These arrays provide a template for RNA molecules that can guide CRISPR-associated (Cas) proteins to specifically neutralize viruses upon subsequent infection. Over the past 10 years, our understanding of CRISPR-Cas systems has benefited greatly from a number of model organisms. In particular, the study of several members of the Gram-negative family, especially and , have provided significant insights into the mechanisms of CRISPR-Cas immunity. In this review, we provide an overview of CRISPR-Cas systems present in members of the . We also detail the current mechanistic understanding of the type I-E and type I-F CRISPR-Cas systems that are commonly found in enterobacteria. Finally, we discuss how phages can escape or inactivate CRISPR-Cas systems and the measures bacteria can enact to counter these types of events.

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Mechanisms of Type I-E and I-F CRISPR-Cas Systems in
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Citation: Xue C, Sashital D. 2019. Mechanisms of Type I-E and I-F CRISPR-Cas Systems in , EcoSal Plus 2019; doi:10.1128/ecosalplus.ESP-0008-2018
/content/ecosalplus/10.1128/ecosalplus.ESP-0008-2018.fig1
Figure 1
CRISPR-Cas operons contain CRISPR arrays and CRISPR-associated () genes. In the adaptation stage, short DNA fragments generated from phage DNA are captured by the adaptation complex. Following trimming of excess DNA, the adaptation complex integrates these fragments into the CRISPR array. During the expression and maturation stage, the CRISPR array is transcribed into long pre-CRISPR RNAs (pre-crRNAs). The pre-crRNAs are cleaved within the repeat regions to generate mature crRNAs. Each crRNA assembles with Cas proteins to form a surveillance complex. During the interference stage, the surveillance complex recognizes the target by complementary base-pairing with the crRNA sequence. Target binding triggers recruitment of a nuclease, which catalyzes degradation of the target nucleic acid.
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Figure 1

CRISPR-Cas operons contain CRISPR arrays and CRISPR-associated () genes. In the adaptation stage, short DNA fragments generated from phage DNA are captured by the adaptation complex. Following trimming of excess DNA, the adaptation complex integrates these fragments into the CRISPR array. During the expression and maturation stage, the CRISPR array is transcribed into long pre-CRISPR RNAs (pre-crRNAs). The pre-crRNAs are cleaved within the repeat regions to generate mature crRNAs. Each crRNA assembles with Cas proteins to form a surveillance complex. During the interference stage, the surveillance complex recognizes the target by complementary base-pairing with the crRNA sequence. Target binding triggers recruitment of a nuclease, which catalyzes degradation of the target nucleic acid.

Citation: Xue C, Sashital D. 2019. Mechanisms of Type I-E and I-F CRISPR-Cas Systems in , EcoSal Plus 2019; doi:10.1128/ecosalplus.ESP-0008-2018
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Figure 2
Type I-E CRISPR-Cas operon of K-12. Promoters are shown as arrows. Repressors of the and promoters are indicated. Type I-F CRISPR-Cas operon of . CRISPR 1 is expressed on the minus strand. Activator of the promoter, which controls expression of all genes, is indicated.
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Figure 2

Type I-E CRISPR-Cas operon of K-12. Promoters are shown as arrows. Repressors of the and promoters are indicated. Type I-F CRISPR-Cas operon of . CRISPR 1 is expressed on the minus strand. Activator of the promoter, which controls expression of all genes, is indicated.

Citation: Xue C, Sashital D. 2019. Mechanisms of Type I-E and I-F CRISPR-Cas Systems in , EcoSal Plus 2019; doi:10.1128/ecosalplus.ESP-0008-2018
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Figure 3
During naive adaptation, RecBCD products serve as substrates for the Cas1-Cas2 adaptation complex (gold). The abundance of Chi sites (magenta) prevents accumulation of DNA fragments from the host genome, while the lack of Chi sites in foreign DNA leads to a larger pool from this source. Replication forks are common sites for DSBs, which are initiation sites for RecBCD degradation sites. Higher-copy foreign DNA contains more replication forks and therefore leads to more DSBs and RecBCD recruitment. Phage DNA is often injected in a linear form, and these ends are subject to degradation by RecBCD. Also shown are two potential mechanisms for primed adaptation: During interference-dependent primed adaptation, the surveillance complex (light blue) directs Cas3 (dark blue) degradation of the foreign DNA. Cas3 products create a DNA fragment pool that provides prespacer substrates for Cas1-Cas2. During interference-independent primed adaptation, both Cas3 and Cas1-Cas2 (or Cas1-Cas2/3) are recruited to the target-bound surveillance complex. The Cas3 helicase domain extrudes a DNA loop as the priming complex searches for prespacer substrates and excises them through the nuclease activity of either Cas1, Cas3, or both subunits.
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Figure 3

During naive adaptation, RecBCD products serve as substrates for the Cas1-Cas2 adaptation complex (gold). The abundance of Chi sites (magenta) prevents accumulation of DNA fragments from the host genome, while the lack of Chi sites in foreign DNA leads to a larger pool from this source. Replication forks are common sites for DSBs, which are initiation sites for RecBCD degradation sites. Higher-copy foreign DNA contains more replication forks and therefore leads to more DSBs and RecBCD recruitment. Phage DNA is often injected in a linear form, and these ends are subject to degradation by RecBCD. Also shown are two potential mechanisms for primed adaptation: During interference-dependent primed adaptation, the surveillance complex (light blue) directs Cas3 (dark blue) degradation of the foreign DNA. Cas3 products create a DNA fragment pool that provides prespacer substrates for Cas1-Cas2. During interference-independent primed adaptation, both Cas3 and Cas1-Cas2 (or Cas1-Cas2/3) are recruited to the target-bound surveillance complex. The Cas3 helicase domain extrudes a DNA loop as the priming complex searches for prespacer substrates and excises them through the nuclease activity of either Cas1, Cas3, or both subunits.

Citation: Xue C, Sashital D. 2019. Mechanisms of Type I-E and I-F CRISPR-Cas Systems in , EcoSal Plus 2019; doi:10.1128/ecosalplus.ESP-0008-2018
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Figure 4
Schematic illustration of Cas1-Cas2 bound to a 23-bp duplex with splayed ends. Individual subunits of Cas1 are shown in orange or gold, individual subunits of Cas2 are shown in light or dark blue, DNA substrate is shown in gray, and PAM is shown in yellow. The 3′-overhangs, one of which contains the PAM, are located in the Cas1 active sites, suggesting that trimming of these ends may be carried out by Cas1. The 5′-overhangs are exposed and may be trimmed by another factor. The trimmed product is a prespacer containing a 23-bp duplex with 5-nt 3′-overhangs containing exposed 3′-hydroxyl groups. Structure of the Cas1-Cas2 complex bound to an untrimmed substrate (PDB number 5DQZ) ( 93 ). Colors are as in panel A. Close-up of Cas1 active site recognition of the 5′-CTT-3′ PAM (yellow) region of the untrimmed prespacer. Hydrogen-bonding interactions are shown with dashed lines. The boxed region shows the scissile phosphate, which is coordinated by the catalytic histidine and aspartate residues of the HD domain. Following cleavage, only the cytosine is retained as the last nucleotide of the trimmed substrate.
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Figure 4

Schematic illustration of Cas1-Cas2 bound to a 23-bp duplex with splayed ends. Individual subunits of Cas1 are shown in orange or gold, individual subunits of Cas2 are shown in light or dark blue, DNA substrate is shown in gray, and PAM is shown in yellow. The 3′-overhangs, one of which contains the PAM, are located in the Cas1 active sites, suggesting that trimming of these ends may be carried out by Cas1. The 5′-overhangs are exposed and may be trimmed by another factor. The trimmed product is a prespacer containing a 23-bp duplex with 5-nt 3′-overhangs containing exposed 3′-hydroxyl groups. Structure of the Cas1-Cas2 complex bound to an untrimmed substrate (PDB number 5DQZ) ( 93 ). Colors are as in panel A. Close-up of Cas1 active site recognition of the 5′-CTT-3′ PAM (yellow) region of the untrimmed prespacer. Hydrogen-bonding interactions are shown with dashed lines. The boxed region shows the scissile phosphate, which is coordinated by the catalytic histidine and aspartate residues of the HD domain. Following cleavage, only the cytosine is retained as the last nucleotide of the trimmed substrate.

Citation: Xue C, Sashital D. 2019. Mechanisms of Type I-E and I-F CRISPR-Cas Systems in , EcoSal Plus 2019; doi:10.1128/ecosalplus.ESP-0008-2018
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Figure 5
Schematic overview of the integration process in . A 3′-OH group of the prespacer attacks the backbone at the leader-proximal end of the first repeat within the CRISPR array, forming a half-site intermediate. The end of the prespacer containing the cytosine from the PAM attacks at the leader-distal end of the opposite strand of the repeat, forming a full-site intermediate. The single-stranded repeats on either end of the integrated spacer are filled by DNA polymerase I, and the nicks are ligated to create an intact CRISPR array. Schematic of leader-proximal and leader-distal integration by Cas1-Cas2 (colored as in Fig. 4 ). Cas1-Cas2 senses the sequence-dependent deformation of the repeat to facilitate integration at the correct sites within the repeat. Cryo-EM structure of Cas1-Cas2 bound to a half-site intermediate containing the IHF-bound leader (PDB number 5WFE) ( 109 ). IHF (dark and light green subunits) binds positions -9 to -35 of the leader (pink) and bends the DNA by ∼180°. This bending brings the upstream binding element (UBS, turquoise) into proximity of the leader-proximal Cas1 dimer, an interaction that ensures integration at the leader-proximal repeat.
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Figure 5

Schematic overview of the integration process in . A 3′-OH group of the prespacer attacks the backbone at the leader-proximal end of the first repeat within the CRISPR array, forming a half-site intermediate. The end of the prespacer containing the cytosine from the PAM attacks at the leader-distal end of the opposite strand of the repeat, forming a full-site intermediate. The single-stranded repeats on either end of the integrated spacer are filled by DNA polymerase I, and the nicks are ligated to create an intact CRISPR array. Schematic of leader-proximal and leader-distal integration by Cas1-Cas2 (colored as in Fig. 4 ). Cas1-Cas2 senses the sequence-dependent deformation of the repeat to facilitate integration at the correct sites within the repeat. Cryo-EM structure of Cas1-Cas2 bound to a half-site intermediate containing the IHF-bound leader (PDB number 5WFE) ( 109 ). IHF (dark and light green subunits) binds positions -9 to -35 of the leader (pink) and bends the DNA by ∼180°. This bending brings the upstream binding element (UBS, turquoise) into proximity of the leader-proximal Cas1 dimer, an interaction that ensures integration at the leader-proximal repeat.

Citation: Xue C, Sashital D. 2019. Mechanisms of Type I-E and I-F CRISPR-Cas Systems in , EcoSal Plus 2019; doi:10.1128/ecosalplus.ESP-0008-2018
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/content/ecosalplus/10.1128/ecosalplus.ESP-0008-2018.fig6
Figure 6
The product of transcription of the CRISPR is a pre-crRNA containing stem-loops within the repeats. Cas6 endoribonucleases (pink) recognize the stem-loops and cleave at the base to form mature crRNAs. Cas6 subunits remain bound to the stem-loop at the 3′-end of the crRNA following cleavage. Six Cas7 subunits (blue) assemble along the length of the crRNA spacer (red), and the Cas5 subunit (purple) caps the Cas7 backbone by specifically recognizing the 5′-repeat-derived handle. In type I-E Cascade, two subtype-specific subunits, Cse2 (orange), assemble on the belly of the complex. The Cas8 subunit (green) caps the complex on the Cas5 end. Schematic of assembled Cascade and Csy complex. Subunits are colored as in panel B, except for Cas8e (panel C) and Cas8f (panel D), which are shown with a green outline. Structures of fully assembled Cascade (PDB ID 4TVX) and Csy (PDB ID 6B45) complex ( 59 , 120 ).
ecosalplus/8/2/ESP-0008-2018_fig_006_thmb.gif
ecosalplus/8/2/ESP-0008-2018_fig_006.gif
Image of Figure 6
Figure 6

The product of transcription of the CRISPR is a pre-crRNA containing stem-loops within the repeats. Cas6 endoribonucleases (pink) recognize the stem-loops and cleave at the base to form mature crRNAs. Cas6 subunits remain bound to the stem-loop at the 3′-end of the crRNA following cleavage. Six Cas7 subunits (blue) assemble along the length of the crRNA spacer (red), and the Cas5 subunit (purple) caps the Cas7 backbone by specifically recognizing the 5′-repeat-derived handle. In type I-E Cascade, two subtype-specific subunits, Cse2 (orange), assemble on the belly of the complex. The Cas8 subunit (green) caps the complex on the Cas5 end. Schematic of assembled Cascade and Csy complex. Subunits are colored as in panel B, except for Cas8e (panel C) and Cas8f (panel D), which are shown with a green outline. Structures of fully assembled Cascade (PDB ID 4TVX) and Csy (PDB ID 6B45) complex ( 59 , 120 ).

Citation: Xue C, Sashital D. 2019. Mechanisms of Type I-E and I-F CRISPR-Cas Systems in , EcoSal Plus 2019; doi:10.1128/ecosalplus.ESP-0008-2018
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Figure 7
Overview of target search by Cascade. Nonspecific interactions at sites with no PAM or PAMs adjacent to nonmatching sequences result in rapid dissociation. At PAM sites, Cascade can initiate unwinding to enable crRNA strand invasion. At partially matching targets, R-loops can form directionally away from the PAM until a mismatch is encountered. Depending on the location and thermodynamic barrier of the mismatch, the R-loop can stall, it can continue to form, or Cascade can dissociate. At a perfect target, complete R-loop formation leads to Cascade locking and a highly stable interaction. Structure of Cascade bound to dsDNA (PDB number 5H9F, colored as in Fig. 6 ) ( 127 ). The Cas11 subunits move down along the belly during R-loop locking. The nontarget strand is stabilized through interactions with Cas8e. Close-up of Cas8e-PAM and Cascade-dsDNA interactions. A glutamine residue acts as a wedge to facilitate DNA unwinding. The PAM is read through minor-groove interactions with Cas8e. Lys-rich loops in Cas7 and Cas7 subunits act as an electrostatic vise to hold the dsDNA during unwinding. The Cas8e CTD four-helix bundle moves toward the N-terminal domain during R-loop locking. Structure of the Csy complex bound to dsDNA (PDB number 6B44, colored as in Fig. 6 ) ( 59 ). The Cas7 backbone elongates by ∼20 Å upon DNA binding. Close-up of Cas8f-PAM and Csy-dsDNA interactions. A lysine residue acts as a wedge to facilitate DNA unwinding. The PAM is read through minor-groove interactions with Cas8f. Several positive residues in Cas7, Cas8f, and Cas5 stabilize interactions with the dsDNA. The Cas8f hook elongates to accommodate the dsDNA.
ecosalplus/8/2/ESP-0008-2018_fig_007_thmb.gif
ecosalplus/8/2/ESP-0008-2018_fig_007.gif
Image of Figure 7
Figure 7

Overview of target search by Cascade. Nonspecific interactions at sites with no PAM or PAMs adjacent to nonmatching sequences result in rapid dissociation. At PAM sites, Cascade can initiate unwinding to enable crRNA strand invasion. At partially matching targets, R-loops can form directionally away from the PAM until a mismatch is encountered. Depending on the location and thermodynamic barrier of the mismatch, the R-loop can stall, it can continue to form, or Cascade can dissociate. At a perfect target, complete R-loop formation leads to Cascade locking and a highly stable interaction. Structure of Cascade bound to dsDNA (PDB number 5H9F, colored as in Fig. 6 ) ( 127 ). The Cas11 subunits move down along the belly during R-loop locking. The nontarget strand is stabilized through interactions with Cas8e. Close-up of Cas8e-PAM and Cascade-dsDNA interactions. A glutamine residue acts as a wedge to facilitate DNA unwinding. The PAM is read through minor-groove interactions with Cas8e. Lys-rich loops in Cas7 and Cas7 subunits act as an electrostatic vise to hold the dsDNA during unwinding. The Cas8e CTD four-helix bundle moves toward the N-terminal domain during R-loop locking. Structure of the Csy complex bound to dsDNA (PDB number 6B44, colored as in Fig. 6 ) ( 59 ). The Cas7 backbone elongates by ∼20 Å upon DNA binding. Close-up of Cas8f-PAM and Csy-dsDNA interactions. A lysine residue acts as a wedge to facilitate DNA unwinding. The PAM is read through minor-groove interactions with Cas8f. Several positive residues in Cas7, Cas8f, and Cas5 stabilize interactions with the dsDNA. The Cas8f hook elongates to accommodate the dsDNA.

Citation: Xue C, Sashital D. 2019. Mechanisms of Type I-E and I-F CRISPR-Cas Systems in , EcoSal Plus 2019; doi:10.1128/ecosalplus.ESP-0008-2018
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Figure 8
Structure of type I-E Cascade-dsDNA following Cas3 recruitment (PDB number 6C66) ( 33 ). Cascade and DNA are colored as in Fig. 6 and 7 ; Cas3 is shown in cyan (helicase and C-terminal domains) and dark blue (HD nuclease domain). Cas3 senses the conformation of Cas8e following R-loop locking. Comparison of Cas8e structure in the locked (PDB number 6C66, green) and unlocked (PDB number 5U07, gray) conformations ( 33 , 128 ). Prior to R-loop locking and movement of the CTD, a loop and helix of the Cas8e CTD would cause steric clashes with the Cas3 nuclease domain. Also shown is a schematic of Cas3 DNA reeling and degradation: Following its recruitment to the R-loop, Cas3 nicks the nontarget strand. The nicked strand is loaded into the Cas3 helicase domain, stabilizing the Cas3-Cascade interaction. The Cas3 helicase domain reels the nontarget strand, while the target strand is extruded and forms an ssDNA loop. The HD nuclease domain can nick the nontarget strand intermittently, creating products that could serve as prespacer substrates for Cas1-Cas2 ( Fig. 3E ), or it can release the DNA and repeat the reeling motion. In type I-F systems, two Cas2/3 molecules may be recruited to the Csy complex, allowing for bidirectional degradation of both strands of the DNA.
ecosalplus/8/2/ESP-0008-2018_fig_008_thmb.gif
ecosalplus/8/2/ESP-0008-2018_fig_008.gif
Image of Figure 8
Figure 8

Structure of type I-E Cascade-dsDNA following Cas3 recruitment (PDB number 6C66) ( 33 ). Cascade and DNA are colored as in Fig. 6 and 7 ; Cas3 is shown in cyan (helicase and C-terminal domains) and dark blue (HD nuclease domain). Cas3 senses the conformation of Cas8e following R-loop locking. Comparison of Cas8e structure in the locked (PDB number 6C66, green) and unlocked (PDB number 5U07, gray) conformations ( 33 , 128 ). Prior to R-loop locking and movement of the CTD, a loop and helix of the Cas8e CTD would cause steric clashes with the Cas3 nuclease domain. Also shown is a schematic of Cas3 DNA reeling and degradation: Following its recruitment to the R-loop, Cas3 nicks the nontarget strand. The nicked strand is loaded into the Cas3 helicase domain, stabilizing the Cas3-Cascade interaction. The Cas3 helicase domain reels the nontarget strand, while the target strand is extruded and forms an ssDNA loop. The HD nuclease domain can nick the nontarget strand intermittently, creating products that could serve as prespacer substrates for Cas1-Cas2 ( Fig. 3E ), or it can release the DNA and repeat the reeling motion. In type I-F systems, two Cas2/3 molecules may be recruited to the Csy complex, allowing for bidirectional degradation of both strands of the DNA.

Citation: Xue C, Sashital D. 2019. Mechanisms of Type I-E and I-F CRISPR-Cas Systems in , EcoSal Plus 2019; doi:10.1128/ecosalplus.ESP-0008-2018
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Tables

Mechanisms of Type I-E and I-F CRISPR-Cas Systems in
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Citation: Xue C, Sashital D. 2019. Mechanisms of Type I-E and I-F CRISPR-Cas Systems in , EcoSal Plus 2019; doi:10.1128/ecosalplus.ESP-0008-2018
/content/ecosalplus/10.1128/ecosalplus.ESP-0008-2018.T1
Table 1
Cas proteins of type I-E and I-F CRISPR-Cas systems
Generic image for table
Table 1

Cas proteins of type I-E and I-F CRISPR-Cas systems

Citation: Xue C, Sashital D. 2019. Mechanisms of Type I-E and I-F CRISPR-Cas Systems in , EcoSal Plus 2019; doi:10.1128/ecosalplus.ESP-0008-2018

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