High-throughput analysis of type I-E CRISPR/Cas spacer acquisition in<i>E. coli</i>

Savitskaya, Ekaterina; Semenova, Ekaterina; Dedkov, Vladimir G.; Metlitskaya, Anastasia; Severinov, Konstantin

doi:10.4161/rna.24325

Cited by 95 publications

(151 citation statements)

References 37 publications

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“…S5). This result corroborates a previous high-throughput spacer analysis from an E. coli plasmid (20). We speculate that this motif was not identified in spacers derived from the plasmid due to the low number of protospacers having a 5′-AAG in the plasmid.…”

Section: Analysis Of Approximately a Million Spacers Shows That The Aamsupporting

confidence: 79%

“…Such a scanning model has been proposed to explain the observed "priming" mechanism, in which acquisition is facilitated from a particular DNA strand if a spacer from that strand is present in the array (15,16). Although a recent study negated the scanning model in a different experimental settings (20), it is possible that it operates on certain DNA segments, under certain conditions. Our data thus corroborate the scanning model, but further studies are required to prove it.…”

Section: Sequence Analysis Of Acquired Spacers Confirms That the Intementioning

confidence: 99%

“…The total number of 5′-AAG on both strands of the pCas1+2 plasmid is 138, and 102 in the plasmid analyzed in ref. 20, whereas such protospacers occur in the chromosome 125,223 times. The analyses are further disturbed by the fact that other dinucleotides at this position may have similar effects as 5′-AA, as shown for one of the tested protospacers (Fig.…”

Section: Analysis Of Approximately a Million Spacers Shows That The Aammentioning

confidence: 99%

“…A recent study used high-throughput sequencing to obtain ∼200,000 spacers derived from plasmids (20). Analysis of the obtained spacers showed that there is a clear preference for acquisition of certain plasmid protospacers over others, all having PAMs.…”

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confidence: 99%

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DNA motifs determining the efficiency of adaptation into the Escherichia coli CRISPR array

Yosef

Shitrit

Goren

et al. 2013

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

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Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins constitute a recently identified prokaryotic defense system against invading nucleic acids. DNA segments, termed protospacers, are integrated into the CRISPR array in a process called adaptation. Here, we establish a PCR-based assay that enables evaluating the adaptation efficiency of specific spacers into the type I-E Escherichia coli CRISPR array. Using this assay, we provide direct evidence that the protospacer adjacent motif along with the first base of the protospacer (5′-AAG) partially affect the efficiency of spacer acquisition. Remarkably, we identified a unique dinucleotide, 5′-AA, positioned at the 3′ end of the spacer, that enhances efficiency of the spacer's acquisition. Insertion of this dinucleotide increased acquisition efficiency of two different spacers. DNA sequencing of newly adapted CRISPR arrays revealed that the position of the newly identified motif with respect to the 5′-AAG is important for affecting acquisition efficiency. Analysis of approximately 1 million spacers showed that this motif is overrepresented in frequently acquired spacers compared with those acquired rarely. Our results represent an example of a short nonprotospacer adjacent motif sequence that affects acquisition efficiency and suggest that other as yet unknown motifs affect acquisition efficiency in other CRISPR systems as well.defense mechanism | phage-host interaction | acquisition step C lustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) comprise an important prokaryotic defense system against horizontally transferred DNA (1-3) and RNA (4). This system shows remarkable analogies to the mammalian immune system (5, 6) and to eukaryotic RNA-interference mechanisms (7, 8). Three major types and 10 subtypes of CRISPR/Cas systems (9) have been found across ∼90% of archaeal genomes and ∼50% of bacterial genomes. All types consist of a CRISPR array-short repeated sequences called "repeats" flanking short sequences called "spacers." The array is usually preceded by a leader, AT-rich DNA sequence that drives CRISPR array expression and is important for acquiring new spacers into the array (10, 11). A cluster of CRISPR-associated (cas) genes encoding proteins that process the transcript, interfere with foreign nucleic acids, and acquire new spacers usually lies adjacent to the CRISPR array (12)(13)(14). RNA transcribed from the CRISPR array (crRNA) is processed by Cas proteins into RNA-based spacers flanked by partial repeats. These crRNAs specifically direct Cas interfering proteins to target nucleic acids matching the spacers. The spacers are acquired from these targeted sequences, termed "protospacers." Spacer acquisition into the CRISPR array consequently results in guiding the system to cleave DNA molecules harboring the corresponding protospacers. This feature renders the system competent in adaptively and specifically targeting invaders.Spacer acquisition into a CRISPR array...

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Section: Analysis Of Approximately a Million Spacers Shows That The Aamsupporting

confidence: 79%

Section: Sequence Analysis Of Acquired Spacers Confirms That the Intementioning

confidence: 99%

Section: Analysis Of Approximately a Million Spacers Shows That The Aammentioning

confidence: 99%

mentioning

confidence: 99%

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DNA motifs determining the efficiency of adaptation into the Escherichia coli CRISPR array

Yosef

Shitrit

Goren

et al. 2013

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

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“…An intact spacer sequence results in interference, while mutation in the protospacer can abolish the CRISPR interfering activity. However, for some systems, when the CRISPR already contains a spacer that matches the invading DNA with a protospacer mismatch in its seed sequence and/or mutation in the PAM, the CRISPR-Cas machinery is used to accelerate the spacer acquisition of the targeted foreign DNA element in a phenomenon called priming (45)(46)(47). In E. coli, the new spacers acquired by priming come from the same strand as the first spacer acquired (25,(45)(46)(47), while in other organisms, this strand bias has not been observed (48,49).…”

Section: Incompatibility Of Plasmids and Crispr-cas Systemsmentioning

confidence: 99%

The CRISPR-Cas Immune System and Genetic Transfers: Reaching an Equilibrium

2015

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Horizontal gene transfer drives the evolution of bacterial genomes, including the adaptation to changing environmental conditions. Exogenous DNA can enter a bacterial cell through transformation (free DNA or plasmids) or through the transfer of mobile genetic elements by conjugation (plasmids) and transduction (bacteriophages). Favorable genes can be acquired, but undesirable traits can also be inadvertently acquired through these processes. Bacteria have systems, such as clustered regularly interspaced short palindromic repeat CRISPR-associated genes (CRISPR-Cas), that can cleave foreign nucleic acid molecules. In this review, we discuss recent advances in understanding CRISPR-Cas system activity against mobile genetic element transfer through transformation and conjugation. We also highlight how CRISPR-Cas systems influence bacterial evolution and how CRISPR-Cas components affect plasmid replication.

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CRISPR Cas

Šviković

2022

Genome Editing in Drug Discovery

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High-throughput analysis of type I-E CRISPR/Cas spacer acquisition inE. coli

Cited by 95 publications

References 37 publications

DNA motifs determining the efficiency of adaptation into the Escherichia coli CRISPR array

DNA motifs determining the efficiency of adaptation into the Escherichia coli CRISPR array

The CRISPR-Cas Immune System and Genetic Transfers: Reaching an Equilibrium

CRISPR Cas

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