Lessons from Enzyme Kinetics Reveal Specificity Principles for RNA-Guided Nucleases in RNA Interference and CRISPR-Based Genome Editing

Bisaria, Namita; Jarmoskaite, Inga; Herschlag, Daniel

doi:10.1016/j.cels.2016.12.010

Cited by 74 publications

(83 citation statements)

References 76 publications

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“…These results indicate that target site unfolding is the rate-limiting step that determines miRISC activity in vivo. Furthermore, this structure-limiting scenario reveals that the ribonuclease AGO undergoes "sticky regime" activation (51), where substrate mRNAs associate and dissociate with AGO more slowly than they are being cleaved.…”

Section: The Unfolding Of the Target Site Structure Is The Primary Dementioning

confidence: 99%

Intact RNA structurome reveals mRNA structure-mediated regulation of miRNA cleavagein vivo

Yang

Woolfenden

Zhang

et al. 2019

Preprint

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MicroRNA (miRNA)-mediated cleavage is involved in numerous essential cellular pathways. miRNAs recognize target RNAs via sequence complementarity. In addition to complementarity, in vitro and in silico studies have suggested that RNA structure may influence the accessibility of mRNAs to miRNA-Induced Silencing Complexes (miRISCs), thereby affecting RNA silencing. However, the regulatory mechanism of mRNA structure in miRNA cleavage remains elusive. Here, we investigated the role of in vivo RNA secondary structure in miRNA cleavage by developing the new CAP-STRUCTURE-seq method to capture the intact mRNA structurome in Arabidopsis thaliana. This approach revealed that miRNA target sites were not structurally accessible for miRISC binding prior to cleavage in vivo. Instead, the unfolding of the target site structure is the primary determinant for miRISC activity in vivo. Notably, we found that the single-strandedness of the two nucleotides immediately downstream of the target site, named Target Adjacent structure Motif (TAM), can promote miRNA cleavage but not miRNA binding, thus decoupling target site binding from cleavage. Our findings demonstrate that mRNA structure in vivo can regulate miRNA cleavage, providing evidence of mRNA structure-dependent regulation of biological processes.

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Section: The Unfolding Of the Target Site Structure Is The Primary Dementioning

confidence: 99%

Intact RNA structurome reveals mRNA structure-mediated regulation of miRNA cleavagein vivo

Yang

Woolfenden

Zhang

et al. 2019

Preprint

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“…Biochemically, we found that these Cas9 variants cleaved the on-target DNA with rates similar to that of wild-type (WT) SpCas9, whereas their cleavage activity was significantly reduced on substrates bearing mismatches (Extended Data Figures 1a, 2a). To test the hypothesis that SpCas9 with its single-guide RNA (sgRNA) might exhibit a greater affinity for its target than is required for effective recognition 9,11 , we measured DNA binding affinity and cleavage of SpCas9-HF1 and eSpCas9(1.1) variants. Contrary to a potential hypothesis that mutating these charged residues to alanine weakens target binding 11 , the affinities of these variants for on-target and PAM-distal mismatched substrates were similar to WT SpCas9 (Figure 1b, Extended Data Figures 1a, 2b), indicating that cleavage specificity is improved through a mechanism distinct from a reduction of target binding affinity 11 .…”

mentioning

confidence: 99%

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy

Chen¹,

Dagdas²,

Kleinstiver³

et al. 2017

Nature

949

877

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The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing1–4. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity were unknown5–9. Using single-molecule Förster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state10 when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we designed a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.

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“…It was argued that this might be the way for bacteria to fight quickly mutating viruses, in which the original target sequence would slightly change (17,28). The probability of such off-target cutting events can reach up to 10−20%, and this significantly complicates the application of CRISPR systems for genetic applications (11).…”

Section: Dynamics Of Search With Off-target Cuttingmentioning

confidence: 99%

“…Despite the tremendous importance of CRISPR-associated processes, there is a surprisingly small number of theoretical investigations concerned with molecular mechanisms and dynamics of underlying processes in these systems (13,27,28). A recent computational study probed structural dynamics of the CRISPR-Cas9 RNA guided DNA cleavage by utilizing various high-resolution structures of Cas9 proteins in different states (13).…”

Section: Introductionmentioning

confidence: 99%

Mechanism of Genome Interrogation: How CRISPR RNA-Guided Cas9 Proteins Locate Specific Targets on DNA

Shvets

Kolomeisky

2017

Biophysical Journal

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The ability to precisely edit and modify genome opens endless opportunities to investigate fundamental properties of living systems as well as to advance various medical techniques and bioengineering applications. This possibility is now close to reality due to a recent discovery of the adaptive bacterial immune system, which is based on clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas) that utilize RNA to find and cut the double-stranded DNA molecules at specific locations. Here we develop a quantitative theoretical approach to analyze the mechanism of target search on DNA by CRISPR RNA-guided Cas9 proteins, which is followed by a selective cleavage of nucleic acids. It is based on a discrete-state stochastic model that takes into account the most relevant physical-chemical processes in the system. Using a method of first-passage processes, a full dynamic description of the target search is presented. It is found that the location of specific sites on DNA by CRISPR Cas9 proteins is governed by binding first to protospacer adjacent motif (PAM) sequences on DNA, which is followed by reversible transitions into DNA interrogation states. In addition, the search dynamics is strongly influenced by the off-target cutting. Our theoretical calculations allows us to explain the experimental observations and to give experimentally testable predictions. Thus, the presented theoretical model clarifies some molecular aspects of the genome interrogation by CRISPR RNA-guided Cas9 proteins.

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Lessons from Enzyme Kinetics Reveal Specificity Principles for RNA-Guided Nucleases in RNA Interference and CRISPR-Based Genome Editing

Cited by 74 publications

References 76 publications

Intact RNA structurome reveals mRNA structure-mediated regulation of miRNA cleavagein vivo

Intact RNA structurome reveals mRNA structure-mediated regulation of miRNA cleavagein vivo

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy

Mechanism of Genome Interrogation: How CRISPR RNA-Guided Cas9 Proteins Locate Specific Targets on DNA

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