Altered stoichiometry<i>Escherichia coli</i>Cascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation

Kuznedelov, Konstantin; Mekler, Vladimir; Lemak, Sofia; Tokmina‐Lukaszewska, Monika; Datsenko, Kirill A.; Jain, Ishita; Savitskaya, Ekaterina; Mallon, John; Shmakov, Sergey; Bothner, Brian; Bailey, Scott; Yakunin, Alexander F.; Severinov, Konstantin; Semenova, Ekaterina

doi:10.1093/nar/gkw914

Cited by 41 publications

(51 citation statements)

References 40 publications

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“…We also found that different lengths of crRNAs corresponded to different numbers of backbone (Csm3) and belly (Csm2) subunits in the complex, consistent with the idea that these subunits assemble along the length of the crRNA. The length of Type I Cascade complexes can also be altered by changing the length of the crRNA, suggesting a conserved mechanism for regulation of complex stoichiometry between Type I and III systems [37–39]. …”

Section: Discussionmentioning

confidence: 99%

RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System

2017

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CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are RNA-guided adaptive immunity pathways used by bacteria and archaea to defend against phages and plasmids. Type III-A systems use a multisubunit interference complex called Csm, containing Cas proteins and a CRISPR RNA (crRNA) to target cognate nucleic acids. The Csm complex is intriguing in that it mediates RNA-guided targeting of both RNA and transcriptionally active DNA, but the mechanism is not well understood. Here, we overexpressed the five components of the Thermus thermophilus (T. thermophilus) Type III-A Csm complex (TthCsm) with a defined crRNA sequence, and purified intact TthCsm complexes from E. coli cells. The complexes were thermophilic, targeting complementary ssRNA more efficiently at 65°C than at 37°C. Sequence-independent, endonucleolytic cleavage of single-stranded DNA (ssDNA) by TthCsm was triggered by recognition of a complementary ssRNA, and required a lack of complementarity between the first 8 nucleotides (5′ tag) of the crRNA and the 3′ flanking region of the ssRNA. Mutation of the histidine-aspartate (HD) nuclease domain of the TthCsm subunit, Cas10/Csm1, abolished DNA cleavage. Activation of DNA cleavage was dependent on RNA binding but not cleavage. This leads to a model in which binding of an ssRNA target to the Csm complex would stimulate cleavage of exposed ssDNA in the cell, such as could occur when the RNA polymerase unwinds double-stranded DNA (dsDNA) during transcription. Our findings establish an amenable, thermostable system for more in-depth investigation of the targeting mechanism using structural biology methods, such as cryo-electron microscopy and x-ray crystallography.

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Section: Discussionmentioning

confidence: 99%

RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System

2017

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“…Cas9 binding to a beacon results in a readily detectable increase in fluorescence intensity. The rate of fluorescence increase depends on the beacon sequence and structure, which can be varied easily for particular applications (19,20). Schematic representations of the beacon assay and structures of Cas9 beacons 1 and 2 used in this work are shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation

Mekler

Minakhin

Severinov

2017

Proc. Natl. Acad. Sci. U.S.A.

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The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves doublestranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA-DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/ single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/ guide RNA-DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs.Cas9 | CRISPR | DNA interrogation | protein-DNA interactions | fluorescence spectroscopy P rokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) gene systems acquire fragments of foreign DNA (protospacers) and insert them as spacers into the CRISPR array in the prokaryotic host genome. Upon subsequent encounters, the complex of Cas proteins with CRISPR RNA (crRNA) bearing the spacer sequence binds and cleaves foreign DNA containing the matching sequence (1-3), thus providing the host organism with adaptive hereditable immunity. The DNA endonuclease Cas9 of type II CRISPR-Cas systems is targeted to specific DNA sequences by a 20-base crRNA spacer that binds to the complementary strand of protospacer DNA, displacing the noncomplementary strand to form an R-loop (4, 5). Both binding and cleavage of target DNA by the Cas9/crRNA complex require a short protospacer adjacent motif (PAM) located immediately downstream of the targeted sequence (6). For the most commonly used Streptococcus pyogenes Cas9 (SpCas9), the PAM sequence is 5′-NGG (7). The perfect match between guide RNA and seven to 12 bases of target DNA at the immediate 5′ side of PAM ("seed" region) is the most critical for DNA binding and cleavage, although limited mispairing in the distal bases is tolerated (8). Two RNA molecules (crRNA and a transactivating crRNA (tracrRNA)) required to guide SpCas9 to targets can be replaced with...

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“…These plasmids were introduced into a strain of E. coli that expresses the full set of type I-E Cas genes required for adaptation and defence (BW40114) 24 . First, we used these strains to see whether any of the cloned plasmid-based NCAs could function in direct interference through a plasmid interference assay 25 by attempting to transform an additional plasmid that also contained the M13 spacer target sequence into cultures expressing the NCA and Cas proteins. If the NCAs are functional, they should reduce the efficiency of this transformation relative to a negative-control plasmid that does not contain a matching protospacer target.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, it was shown that a more sensitive in vivo test of crRNA function is a primed acquisition assay 25 . Briefly, ‘priming’ is the efficient acquisition of new spacers during a phage challenge of this system stimulated by a pre-existing spacer matching the phage genome that enhances the acquisition of additional phage spacers.…”

Section: Resultsmentioning

confidence: 99%

Spontaneous CRISPR loci generation in vivo by non-canonical spacer integration

2018

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The adaptation phase of CRISPR-Cas immunity depends on the precise integration of short segments of foreign DNA (spacers) into a specific genomic location within the CRISPR locus by the Cas1-Cas2 integration complex. Although off-target spacer integration outside of canonical CRISPR arrays has been described in vitro, no evidence of non-specific integration activity has been found in vivo. Here, we show that non-canonical off-target integrations can occur within bacterial chromosomes at locations that resemble the native CRISPR locus by characterizing hundreds of off-target integration locations within Escherichia coli. Considering whether such promiscuous Cas1-Cas2 activity could have an evolutionary role through the genesis of neo-CRISPR loci, we combed existing CRISPR databases and available genomes for evidence of off-target integration activity. This search uncovered several putative instances of naturally occurring off-target spacer integration events within the genomes of Yersinia pestis and Sulfolobus islandicus. These results are important in understanding alternative routes to CRISPR array genesis and evolution, as well as in the use of spacer acquisition in technological applications.

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Altered stoichiometryEscherichia coliCascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation

Cited by 41 publications

References 40 publications

RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System

RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System

Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation

Spontaneous CRISPR loci generation in vivo by non-canonical spacer integration

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