8 research outputs found

    Steady-state levels of PRV•CR and PRV•M transcripts in the presence or in the absence of C

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    <p><b>Copyright information:</b></p><p>Taken from "Transcription regulation of the EcoRV restriction–modification system"</p><p>Nucleic Acids Research 2005;33(21):6942-6951.</p><p>Published online 6 Dec 2005</p><p>PMCID:PMC1310966.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p>EcoRV. RNA was purified from the HB101 cells harboring the indicated plasmids and primer extension reactions were performed to reveal 5′ end of divergent RNAs arising from the EcoRV regulatory region. The R primer allows the detection of rightward transcripts from pR or pM (PRV•CR or PRV•M transcripts, correspondingly). The L primer allows the detection of leftward transcripts from pR or pM (PRV•M or PRV•CR transcripts, correspondingly). The sequencing reactions' marker lanes were prepared using the pM or pR plasmids and primers used for primer extension

    Defining the seed sequence of the Cas12b CRISPR-Cas effector complex

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    <p>Target binding by CRISPR-Cas ribonucleoprotein effectors is initiated by the recognition of double-stranded PAM motifs by the Cas protein moiety followed by destabilization, localized melting, and interrogation of the target by the guide part of CRISPR RNA moiety. The latter process depends on seed sequences, parts of the target that must be strictly complementary to CRISPR RNA guide. Mismatches between the target and CRISPR RNA guide outside the seed have minor effects on target binding, thus contributing to off-target activity of CRISPR-Cas effectors. Here, we define the seed sequence of the Type V Cas12b effector from <i>Bacillus thermoamylovorans</i>. While the Cas12b seed is just five bases long, in contrast to all other effectors characterized to date, the nucleotide base at the site of target cleavage makes a very strong contribution to target binding. The generality of this additional requirement was confirmed during analysis of target recognition by Cas12b effector from <i>Alicyclobacillus acidoterrestris</i>. Thus, while the short seed may contribute to Cas12b promiscuity, the additional specificity determinant at the site of cleavage may have a compensatory effect making Cas12b suitable for specialized genome editing applications.</p

    Type I-E CRISPR-Cas Systems Discriminate Target from Non-Target DNA through Base Pairing-Independent PAM Recognition

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    <div><p>Discriminating self and non-self is a universal requirement of immune systems. Adaptive immune systems in prokaryotes are centered around repetitive loci called CRISPRs (clustered regularly interspaced short palindromic repeat), into which invader DNA fragments are incorporated. CRISPR transcripts are processed into small RNAs that guide CRISPR-associated (Cas) proteins to invading nucleic acids by complementary base pairing. However, to avoid autoimmunity it is essential that these RNA-guides exclusively target invading DNA and not complementary DNA sequences (i.e., self-sequences) located in the host's own CRISPR locus. Previous work on the Type III-A CRISPR system from <i>Staphylococcus epidermidis</i> has demonstrated that a portion of the CRISPR RNA-guide sequence is involved in self versus non-self discrimination. This self-avoidance mechanism relies on sensing base pairing between the RNA-guide and sequences flanking the target DNA. To determine if the RNA-guide participates in self versus non-self discrimination in the Type I-E system from <i>Escherichia coli</i> we altered base pairing potential between the RNA-guide and the flanks of DNA targets. Here we demonstrate that Type I-E systems discriminate self from non-self through a base pairing-independent mechanism that strictly relies on the recognition of four unchangeable PAM sequences. In addition, this work reveals that the first base pair between the guide RNA and the PAM nucleotide immediately flanking the target sequence can be disrupted without affecting the interference phenotype. Remarkably, this indicates that base pairing at this position is not involved in foreign DNA recognition. Results in this paper reveal that the Type I-E mechanism of avoiding self sequences and preventing autoimmunity is fundamentally different from that employed by Type III-A systems. We propose the exclusive targeting of PAM-flanked sequences to be termed a target versus non-target discrimination mechanism.</p></div

    Synonymous mutations of the crRNA and the PAM do not affect self versus non-self discrimination.

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    <p><b>A</b>) Infectivity of a library of M13 phage containing PAM mutants adjacent to the g8 protospacer was tested against cells expressing Cascade, Cas3 and g8 CRISPR containing mutations at the −1, −2 and −3 nucleotides of the CRISPR repeats. PAM mutations are shown on the left, with the PAM sequences indicated in the 3′ to 5′ direction. CRISPR repeat mutations at positions −1, −2 and −3 are indicated on the top in the 5′ to 3′ direction. Underscored sequences have been tested for binding affinity by EMSA. Base pairing potential between the PAM positions and the repeat is indicated using numbers (0–7) that correspond to a base pairing pattern that is shown in the panel on the right. A 0 signifies no base pairing, a 1 signifies base pairing at the −3 position, a 2 signifies base pairing at the −2 position, etc. Black circles with white digits indicate resistance against phage infection (e.o.p.<10<sup>−4</sup>), grey circles indicate partial resistance (e.o.p.∼10<sup>−2</sup>) and red digits without circle indicate susceptibility to phage infection (e.o.p. = 1), as determined by phage spot assays. Letters B, C, D, E indicate combinations shown in detail in the corresponding panels. <b>B</b>) Combination of g8<sup>C-3AC-2T</sup> CRISPR and M13 phage with CAT PAM, gives rise to full base pairing and a lack of resistance (red font). <b>C</b>) Combination of g8<sup>C-3AC-2T</sup> CRISPR and M13 phage with CTC PAM, gives rise to only base pairing at the −1 position and yields a lack of resistance (red font). <b>D</b>) Combination of g8<sup>C-3AC-2A</sup> CRISPR and M13 phage with CTT PAM gives rise to full base pairing and a lack of resistance (red font). <b>E</b>) Combination of g8<sup>C-3AC-2A</sup> CRISPR and M13 phage with CTC PAM gives rise to only two potential base pairs at the −1 and −2 positions and yields a lack of resistance (red font). Note in (A–E) PAMs are oriented in 3′ to 5′direction to display base pairing potential with the last three nucleotides of the crRNA repeat.</p

    Potential base pairing between the crRNA repeat regions and protospacer flanking regions does not affect CRISPR-interference.

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    <p><b>A</b>) Model of the R-loop formed by Cascade during dsDNA binding. <b>B</b>) Cells expressing WT g8-Cascade and Cas3 are resistant to plasmids containing the CAT PAM adjacent to the g8 protospacer (black bars, transformation efficiency 6.7±1.5×10<sup>5</sup> cfu/µg DNA for plasmid pWUR690 and 6.8±0.9×10<sup>5</sup> cfu/µg DNA for plasmid pWUR688), but are susceptible to plasmid transformation when the g8 protospacer is flanked by a CGG PAM, which is fully complementary to the 5′-handle (red bars, transformation efficiency 4.2±0.9×10<sup>8</sup> cfu/µg DNA for plasmid pWUR687 and 4.5±0.8×10<sup>8</sup> cfu/µg DNA for plasmid pWUR689). Transformation efficiency for a control pUC19 plasmid is 6.2±1.1×10<sup>8</sup> cfu/µg DNA. The histogram shows the <i>in vitro</i> binding affinity of purified WT g8-Cascade for dsDNA containing the g8 protospacer flanked by sequences with a varying base pairing potential, as shown on the right. Asterisks indicate that the Kd value is >>1000 nM and the error bars represent the standard deviation of the mean.</p

    Base pairing at the −2 and −3 positions does not interfere with CRISPR-immunity.

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    <p><b>A</b>) Cells expressing g8<sup>C-2A</sup>-Cascade and Cas3 are partially resistant to M13 phage containing the CTT PAM adjacent to the g8 protospacer (white font/grey bar, e.o.p.∼10<sup>−2</sup>), but not when containing the CAT, CCT, CTC or CGT PAM (red font/red bars). Note that in the figure the PAMs are oriented in 3′ to 5′direction to display base pairing potential with the last three nucleotides of the crRNA repeat. The <i>in vitro</i> binding affinity of purified WT g8<sup>C-2A</sup>-Cascade for dsDNA containing the g8 protospacer and each of the respective PAM mutants is shown in the adjacent histogram. <b>B</b>) Assays as in (A) using cells expressing the g8<sup>C-3G</sup> CRISPR, Cascade and Cas3, show that cells are not resistant to M13 phage containing the CAT, CTT, CCT, CTC or CGT PAMs adjacent to the g8 protospacer (red font/red bars). The <i>in vitro</i> binding affinity of purified WT g8<sup>C-3G</sup>-Cascade for dsDNA containing the M13 protospacer and each of the respective PAM mutants is shown in the adjacent histogram. Asterisks indicate that the Kd value is >>1000 nM and the error bars represent the standard deviation of the mean.</p

    Base pairing at the −1 position is not required for CRISPR-interference.

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    <p><b>A</b>) Model of the R-loop formed by Cascade during dsDNA binding. The nucleotide adjacent to the spacer sequence (the −1 position) has the potential to base pair with the first nucleotide of the PAM in the target strand of the DNA. <b>B</b>) Cells expressing WT g8-Cascade and Cas3 are resistant to M13 phage containing the CAT, CTT, CCT or CTC PAMs adjacent to the M13 protospacer (white font/black bars, e.o.p.<10<sup>−4</sup>), but not when containing the CGT PAM (red font/red bars, e.o.p. = 1). Note that in the figure the PAMs are oriented in 3′ to 5′direction to display base pairing potential with the last three nucleotides of the crRNA repeat. The <i>in vitro</i> binding affinity of purified WT g8-Cascade for dsDNA containing the g8 protospacer and each of the respective PAM mutants is shown in the adjacent histogram. <b>C</b>) Assays as in (B) using cells expressing the g8<sup>G-1T</sup> CRISPR, Cascade and Cas3, show that cells are resistant to M13 phage containing the CAT, CTT, CCT or CTC PAMs adjacent to the g8 protospacer (white font/black bars), but not when containing the CGT PAM (red font/red bars). The <i>in vitro</i> binding affinity of purified WT g8<sup>G-1T</sup>-Cascade for dsDNA containing the g8 protospacer and each of the respective PAM mutants is shown in the adjacent histogram. In (B) and (C) error bars represent the standard deviation of the mean.</p

    Model of self versus non-self discrimination by Type III-A systems and target versus non-target discrimination by Type I-E systems.

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    <p><b>A</b>) Model of the mechanism employed by Type III-A systems. Type III-A systems presumably target DNA through R-loop formation. The crRNA (here depicted as the 37 nt species) could be part of a ribonucleoprotein complex consisting of Cas proteins and a single crRNA (Csm complex). R-loop formation and subsequent interference is inhibited when base pairing occurs between the −2, −3 and −4 positions of the repeat sequence at the 5′ end of the crRNA and the sequence flanking the 3′ end of the protospacer. How this base pairing is monitored is currently unknown, but it might involve a Cas protein. <b>B</b>) Model of target versus non-target discrimination by Type I-E systems. Type I-E systems target DNA through R-loop formation. The 61 nt crRNA is part of the Cascade ribonucleoprotein complex. R-loop formation and subsequent Cas3-mediated cleavage of the target DNA are activated when a PAM is present at the −1, −2 and −3 positions, in the sequence flanking the 3′ end of the protospacer. The presence of a PAM is monitored through the Cse1 subunit.</p
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