Spontaneous Embedding of DNA Mismatches Within the RNA:DNA Hybrid of CRISPR-Cas9

Mitchell, Brandon P.; Hsu, Rohaine V.; Medrano, Marco A.; Zewde, Nehemiah T.; Narkhede, Yogesh B.; Palermo, Giulia

doi:10.3389/fmolb.2020.00039

Cited by 30 publications

(46 citation statements)

References 41 publications

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“…This theoretical structure enabled to initiate in-depth studies of the catalysis ( Palermo, 2019 ; Casalino et al, 2020 ), the allostery ( Palermo et al, 2017 ; East et al, 2020 ; Nierzwicki et al, 2020 ) and the system’s specificity ( Mitchell et al, 2020 ; Ricci et al, 2019 ), when no structural information on the active state was available. This helped obtaining information to improve the enzyme catalytic efficiency and to reduce off-target effects, which is a key goal for biomedical applications ( Fu et al, 2013 ).…”

Section: Capturing Transitions and Short-lived Conformational Statesmentioning

confidence: 99%

Molecular Dynamics to Predict Cryo-EM: Capturing Transitions and Short-Lived Conformational States of Biomolecules

Nierzwicki

Palermo

2021

Front. Mol. Biosci.

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Single-particle cryogenic electron microscopy (cryo-EM) has revolutionized the field of the structural biology, providing an access to the atomic resolution structures of large biomolecular complexes in their near-native environment. Today’s cryo-EM maps can frequently reach the atomic-level resolution, while often containing a range of resolutions, with conformationally variable regions obtained at 6 Å or worse. Low resolution density maps obtained for protein flexible domains, as well as the ensemble of coexisting conformational states arising from cryo-EM, poses new challenges and opportunities for Molecular Dynamics (MD) simulations. With the ability to describe the biomolecular dynamics at the atomic level, MD can extend the capabilities of cryo-EM, capturing the conformational variability and predicting biologically relevant short-lived conformational states. Here, we report about the state-of-the-art MD procedures that are currently used to refine, reconstruct and interpret cryo-EM maps. We show the capability of MD to predict short-lived conformational states, finding remarkable confirmation by cryo-EM structures subsequently solved. This has been the case of the CRISPR-Cas9 genome editing machinery, whose catalytically active structure has been predicted through both long-time scale MD and enhanced sampling techniques 2 years earlier than cryo-EM. In summary, this contribution remarks the ability of MD to complement cryo-EM, describing conformational landscapes and relating structural transitions to function, ultimately discerning relevant short-lived conformational states and providing mechanistic knowledge of biological function.

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Section: Capturing Transitions and Short-lived Conformational Statesmentioning

confidence: 99%

Molecular Dynamics to Predict Cryo-EM: Capturing Transitions and Short-Lived Conformational States of Biomolecules

Nierzwicki

Palermo

2021

Front. Mol. Biosci.

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“…This clarified how REC3 could act as an allosteric regulator of HNH by “sensing” the RNA:DNA hybrid, as hypothesized previously 17,18 . MD studies also focused on the effect of DNA mismatches within the RNA:DNA heteroduplex, reaching upstream positions with respect to PAM distal ends 64 . Couples of base‐pair mismatches were introduced starting from the PAM distal ends up to the middle of the 20 base pairs RNA:DNA structure (i.e., at position 10).…”

Section: Role Of Allostery In the Onset Of Off‐target Effectsmentioning

confidence: 61%

“…We show that allostery intervenes during at least three steps of the CRISPR-Cas9 function, affecting DNA recognition, 13,15 mediating the cleavage 16,17 and interfering with the off-target activity. 18,19,64 At first, the binding of the PAM recognition sequence was found not only to be necessary to initiate DNA unwinding, but is also critical for the activation of the distally spaced nuclease domains HNH and RuvC. 13,15 The concerted DNA cleavages governed by this PAM-mediated allosteric signaling were found to pass through the L1/L2 loops, which act as "allosteric transducers" that connect the HNH and RuvC catalytic domains.…”

Section: Discussionmentioning

confidence: 99%

“…17,18 MD studies also focused on the effect of DNA mismatches within the RNA:DNA heteroduplex, reaching upstream positions with respect to PAM distal ends. 64 Couples of base-pair mismatches were introduced starting from the PAM distal ends up to the middle of the 20 base pairs RNA:DNA structure (i.e., at position 10). The simulations revealed that mismatched pairs at upstream positions (i.e., from position 10-14) are easily incorporated within the hybrid structure, with minor effect on the protein-nucleic acid interactions.…”

Section: Role Of Allostery In the Onset Of Off-target Effectsmentioning

confidence: 99%

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Establishing the allosteric mechanism in CRISPR‐Cas9

Nierzwicki

Arantes

Saha

et al. 2020

WIREs Comput Mol Sci

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Allostery is a fundamental property of proteins, which regulates biochemical information transfer between spatially distant sites. Here, we report on the critical role of molecular dynamics (MD) simulations in discovering the mechanism of allosteric communication within CRISPR‐Cas9, a leading genome editing machinery with enormous promises for medicine and biotechnology. MD revealed how allostery intervenes during at least three steps of the CRISPR‐Cas9 function: affecting DNA recognition, mediating the cleavage and interfering with the off‐target activity. An allosteric communication that activates concerted DNA cleavages was found to led through the L1/L2 loops, which connect the HNH and RuvC catalytic domains. The identification of these “allosteric transducers” inspired the development of novel variants of the Cas9 protein with improved specificity, opening a new avenue for controlling the CRISPR‐Cas9 activity. Discussed studies also highlight the critical role of the recognition lobe in the conformational activation of the catalytic HNH domain. Specifically, the REC3 region was found to modulate the dynamics of HNH by sensing the formation of the RNA:DNA hybrid. The role of REC3 was revealed to be particularly relevant in the presence of DNA mismatches. Indeed, interference of REC3 with the RNA:DNA hybrid containing mismatched pairs at specific positions resulted in locking HNH in an inactive “conformational checkpoint” conformation, thereby hampering off‐target cleavages. Overall, MD simulations established the fundamental mechanisms underlying the allosterism of CRISPR‐Cas9, aiding engineering strategies to develop new CRISPR‐Cas9 variants for improved genome editing. This article is categorized under: Structure and Mechanism > Computational Biochemistry and Biophysics

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“…In addition, a number of biophysical experiments highlighted that not only PAM proximal but also PAM distal mismatches can strongly inhibit off-target cleavage. The proposed mechanism for this intriguing phenomenon is a balance shift in the HNH domain toward an inactive state (Chen et al, 2017;Dagdas et al, 2017;Ricci et al, 2019;Mitchell et al, 2020). After binding of the target DNA and establishment of the RNA:DNA hybrid at the on-target, the HNH domain flips from an RNA-bound state to an inactive state and then rapidly into a docked-state that enables cleavage (Dagdas et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Which Factors Affect the Occurrence of Off-Target Effects Caused by the Use of CRISPR/Cas: A Systematic Review in Plants

et al. 2020

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CRISPR/Cas enables a targeted modification of DNA sequences. Despite their ease and efficient use, one limitation is the potential occurrence of associated off-target effects. This systematic review aims to answer the following research question: Which factors affect the occurrence of off-target effects caused by the use of CRISPR/Cas in plants? Literature published until March 2019 was considered for this review. Articles were screened for relevance based on pre-defined inclusion criteria. Relevant studies were subject to critical appraisal. All studies included in the systematic review were synthesized in a narrative report, but studies rated as high and medium/high validity were reported separately from studies rated as low and medium/low or unclear validity. In addition, we ran a binary logistic regression analysis to verify five factors that may affect the occurrence of off-target effects: (1) Number of mismatches (2) Position of mismatches (3) GC-content of the targeting sequence (4) Altered nuclease variants (5) Delivery methods. In total, 180 relevant articles were included in this review containing 468 studies therein. Seventy nine percentage of these studies were rated as having high or medium/high validity. Within these studies, 6,416 potential off-target sequences were assessed for the occurrence of off-target effects. Results clearly indicate that an increased number of mismatches between the on-target and potential off-target sequence steeply decreases the likelihood of off-target effects. The observed rate of off-target effects decreased from 59% when there is one mismatch between the on-target and off-target sequences toward 0% when four or more mismatches exist. In addition, mismatch/es located within the first eight nucleotides proximal to the PAM significantly decreased the occurrence of off-target effects. There is no evidence that the GC-content significantly affects off-target effects. The database regarding the impact of the nuclease variant and the delivery method is very poor as the majority of studies applied the standard nuclease SpCas9 and the CRISPR/Cas system was stably delivered in the genome. Hence, a general significant impact of these two factors on the occurrence of off-target effects cannot be proved. This identified evidence gap needs to be filled by systematic studies exploring these individual factors in sufficient numbers.

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Spontaneous Embedding of DNA Mismatches Within the RNA:DNA Hybrid of CRISPR-Cas9

Cited by 30 publications

References 41 publications

Molecular Dynamics to Predict Cryo-EM: Capturing Transitions and Short-Lived Conformational States of Biomolecules

Molecular Dynamics to Predict Cryo-EM: Capturing Transitions and Short-Lived Conformational States of Biomolecules

Establishing the allosteric mechanism in CRISPR‐Cas9

Which Factors Affect the Occurrence of Off-Target Effects Caused by the Use of CRISPR/Cas: A Systematic Review in Plants

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