Planting the seed: target recognition of short guide RNAs

Künne, Tim; Swarts, Daan C.; Brouns, Stan J. J.

doi:10.1016/j.tim.2013.12.003

Cited by 71 publications

(54 citation statements)

References 108 publications

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“…To date, the PAM has been well characterized in a number of type I and type II systems and the effect of mutations in the protospacer has been documented (5,14,23,46,47). However, only few high-throughput random-mutagenesis studies of the effects of PAM and protospacer mutations have been reported.…”

Section: Discussionmentioning

confidence: 99%

Degenerate target sites mediate rapid primed CRISPR adaptation

Fineran

Gerritzen

Suárez-Diez

et al. 2014

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

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Prokaryotes encode adaptive immune systems, called CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated), to provide resistance against mobile invaders, such as viruses and plasmids. Host immunity is based on incorporation of invader DNA sequences in a memory locus (CRISPR), the formation of guide RNAs from this locus, and the degradation of cognate invader DNA (protospacer). Invaders can escape type I-E CRISPRCas immunity in Escherichia coli K12 by making point mutations in the seed region of the protospacer or its adjacent motif (PAM), but hosts quickly restore immunity by integrating new spacers in a positive-feedback process termed "priming." Here, by using a randomized protospacer and PAM library and high-throughput plasmid loss assays, we provide a systematic analysis of the constraints of both direct interference and subsequent priming in E. coli. We have defined a high-resolution genetic map of direct interference by Cascade and Cas3, which includes five positions of the protospacer at 6-nt intervals that readily tolerate mutations. Importantly, we show that priming is an extremely robust process capable of using degenerate target regions, with up to 13 mutations throughout the PAM and protospacer region. Priming is influenced by the number of mismatches, their position, and is nucleotide dependent. Our findings imply that even outdated spacers containing many mismatches can induce a rapid primed CRISPR response against diversified or related invaders, giving microbes an advantage in the coevolutionary arms race with their invaders.adaptive immunity | phage resistance | crRNA | next-generation sequencing | horizontal gene transfer

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Section: Discussionmentioning

confidence: 99%

Degenerate target sites mediate rapid primed CRISPR adaptation

Fineran

Gerritzen

Suárez-Diez

et al. 2014

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

Self Cite

240

365

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“…This preordering is thought to be thermodynamically favorable for target binding, reminiscent of the guide RNA positioning observed in other small regulatory RNA pathways, including the bacterial Hfq protein-RNA complex and eukaryotic Argonaute-mediated RNA silencing (57). Notably, in the type I CRISPR interference complex known as Cascade, the guide RNA is preordered throughout the entire crRNA rather than just in the seed region, likely because of the helical assembly of the complex and release of topological constraints by completely flippedout nucleotides at every sixth position (43,72,110).…”

Section: Preordered Seed Rna and Pam-interacting Cleftmentioning

confidence: 99%

CRISPR–Cas9 Structures and Mechanisms

Jiang

Doudna

2017

Annu. Rev. Biophys.

1,402

1,206

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Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes. The use of CRISPR-Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)-CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.

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“…It has been proposed that the guide-target base pairing nucleates at the seed region (Brodersen and Voinnet, 2009;Kü nne et al, 2014;Schirle and MacRae, 2012;Schirle et al, 2014). To test the hypothesis, we prepared a series of guide RNAs with dinucleotide mismatches covering the 2-21 base-pairing region ( Figure 2C) and studied how they affected the binding rate.…”

Section: Target Binding Nucleates In the Seed Regionmentioning

confidence: 99%

Human Argonaute 2 Has Diverse Reaction Pathways on Target RNAs

et al. 2015

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Argonaute is a key enzyme of various RNA silencing pathways. We use single-molecule fluorescence measurements to characterize the reaction mechanisms of the core-RISC (RNA-induced silencing complex) composed of human Argonaute 2 and a small RNA. We found that target binding of core-RISC starts at the seed region, resulting in four distinct reaction pathways: target cleavage, transient binding, stable binding, and Argonaute unloading. The target cleavage requires extensive sequence complementarity and dramatically accelerates core-RISC recycling. The stable binding of core-RISC is efficiently established with the seed match only, providing a potential explanation for the seed-match rule of miRNA (microRNA) target selection. Target cleavage on perfect-match targets sensitively depends on RNA sequences, providing an insight into designing more efficient siRNAs (small interfering RNAs).

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Planting the seed: target recognition of short guide RNAs

Cited by 71 publications

References 108 publications

Degenerate target sites mediate rapid primed CRISPR adaptation

Degenerate target sites mediate rapid primed CRISPR adaptation

CRISPR–Cas9 Structures and Mechanisms

Human Argonaute 2 Has Diverse Reaction Pathways on Target RNAs

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