Key role of the REC lobe during CRISPR–Cas9 activation by ‘sensing’, ‘regulating’, and ‘locking’ the catalytic HNH domain

Palermo, Giulia; Chen, Janice S.; Ricci, Clarisse Gravina; Rivalta, Ivan; Jínek, Martin; Batista, Víctor S.; Doudna, Jennifer A.; McCammon, J. Andrew

doi:10.1017/s0033583518000070

Cited by 93 publications

(172 citation statements)

References 64 publications

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“…This resembles the reciprocal dynamical role of REC2 and HNH in Cas9, where REC2 regulates conformational changes of the catalytic HNH domain to allow TS cleavage. 19 , 23 − 25 In light of these observations, the dynamics of REC2 in Cas12a could assist the conformational changes of Nuc and acts as a “regulator” of its function. In support of this hypothesis, single-molecule studies of Cas12a have shown that the REC2 and Nuc domains show the largest conformational rearrangements.…”

Section: Discussionmentioning

confidence: 99%

Molecular Dynamics Reveals a DNA-Induced Dynamic Switch Triggering Activation of CRISPR-Cas12a

Saha

Arantes

Hsu

et al. 2020

J. Chem. Inf. Model.

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CRISPR-Cas12a is a genome-editing system, recently also harnessed for nucleic acid detection, which is promising for the diagnosis of the SARS-CoV-2 coronavirus through the DETECTR technology. Here, a collective ensemble of multimicrosecond molecular dynamics characterizes the key dynamic determinants allowing nucleic acid processing in CRISPR-Cas12a. We show that DNA binding induces a switch in the conformational dynamics of Cas12a, which results in the activation of the peripheral REC2 and Nuc domains to enable cleavage of nucleic acids. The simulations reveal that large-amplitude motions of the Nuc domain could favor the conformational activation of the system toward DNA cleavages. In this process, the REC lobe plays a critical role. Accordingly, the joint dynamics of REC and Nuc shows the tendency to prime the conformational transition of the DNA target strand toward the catalytic site. Most notably, the highly coupled dynamics of the REC2 region and Nuc domain suggests that REC2 could act as a regulator of the Nuc function, similar to what was observed previously for the HNH domain in the CRISPR-associated nuclease Cas9. These mutual domain dynamics could be critical for the nonspecific binding of DNA and thereby for the underlying mechanistic functioning of the DETECTR technology. Considering that REC is a key determinant in the system’s specificity, our findings provide a rational basis for future biophysical studies aimed at characterizing its function in CRISPR-Cas12a. Overall, our outcomes advance our mechanistic understanding of CRISPR-Cas12a and provide grounds for novel engineering efforts to improve genome editing and viral detection.

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Section: Discussionmentioning

confidence: 99%

Molecular Dynamics Reveals a DNA-Induced Dynamic Switch Triggering Activation of CRISPR-Cas12a

Saha

Arantes

Hsu

et al. 2020

J. Chem. Inf. Model.

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“…for understanding the molecular basis of the CRISPR-Cas9 function. (13)(14)(15)(16)(17) Among other studies, our group has successfully applied MD simulations to disclose a mechanism for the conformational activation of Cas9, clarifying the activation process by which the HNH domain is repositioned for cleavage in good agreement with structural and smFRET experiments. (18) Remarkably, MD simulations as well as experimental studies have indicated that the conformational changes underlying the CRISPR-Cas9 function occur over and beyond micro-to-millisecond time scales.…”

Section: Introductionmentioning

confidence: 81%

“…Specifically, the REC3 region, which directly contacts the very end of the hybrid, "senses" the formation of the DNA:RNA hybrid and allows for HNH nuclease activation. (7,13) In order to understand the role of REC3 in the hybrid recognition and in the on-target selectivity, we monitored the interactions established by the RNA:DNA hybrid with REC3 in the simulated systems. In the "unproductive" systems, we observe a significant increase of interactions between the hybrid and REC3 (separated in polar, apolar and charged contributions, Fig.…”

Section: Effect Of Off-target Binding On the Hybrid Recognitionmentioning

confidence: 99%

“…(18) Remarkably, MD simulations as well as experimental studies have indicated that the conformational changes underlying the CRISPR-Cas9 function occur over and beyond micro-to-millisecond time scales. (13,19,20) Such long time scales require the use of computational methods that enable enhanced sampling of the configurational space, such as accelerated MD (aMD) simulations. (21) The aMD method adds a boost potential to certain regions of the potential energy surface, effectively decreasing the energy barriers and thus accelerating transitions between low-energy states.…”

Section: Introductionmentioning

confidence: 99%

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Molecular mechanism of off-target effects in CRISPR-Cas9

Ricci

Chen

Miao

et al. 2018

Preprint

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CRISPR-Cas9 is the state-of-the-art technology for editing and manipulating nucleic acids. However, the occurrence of off-target mutations can limit its applicability. Here, allatom enhanced molecular dynamics (MD) simulations -using Gaussian accelerated MD (GaMD) -are used to decipher the mechanism of off-target binding at the molecular level.GaMD reveals that base pair mismatches in the target DNA at specific distal sites with respect to the Protospacer Adjacent Motif (PAM) induce an extended opening of the RNA:DNA heteroduplex, which leads to newly discovered interactions between the unwound nucleic acids and the protein counterpart. The conserved interactions between the target DNA strand and the L2 loop of the catalytic HNH domain constitute a "lock" effectively decreasing the conformational freedom of the HNH domain and its activation for cleavage. Remarkably, depending on their position at PAM distal sites, DNA mismatches leading to off-target cleavages are unable to "lock" the HNH domain, thereby identifying the ability to "lock" HNH as a key determinant. Consistently, off-target sequences hampering the catalysis have been shown to "trap" somehow the HNH domain in an inactive "conformational checkpoint" state (Dagdas et al. Sci Adv, 2017). As such, this mechanism identifies the molecular basis underlying off-target cleavages and contributes in clarifying a long-lasting open issue of the CRISPR-Cas9 function. It also poses the foundation for designing novel and more specific Cas9 variants, which could be obtained by magnifying the "locking" interactions between HNH and the target DNA in the presence of any incorrect off-target sequence, thus preventing undesired cleavages.In this respect, a promising strategy to fight off-target cleavages is the molecular engineering of highly specific Cas9 proteins,(6-8) which would enable safer and easy-to-use applications of the CRISPR technology on a genome-wide scale.(9) However, rational design of CRISPR-Cas9 requires detailed knowledge of the molecular bases underlying off-target effects, which are not yet understood. (10, 11) At the molecular level, off-target effects are the unselective cleavages of DNA sequences that do not fully match the guide RNA, bearing base pair mismatches at PAM distal sites of the DNA:RNA hybrid (Fig. 1). Kinetic and single molecule (sm) Förster Resonance Energy Transfer (FRET) experiments have shown that the occurrence of off-target cleavages is directly related to the conformational state adopted by the catalytic HNH domain.(7, 12) Upon DNA binding, HNH undergoes a conformational change from an inactive state, in which the catalytic H840 is far away from the cleavage site on the TS, to an activated state that is prone for catalysis (Fig. 1A). The inactive state of the HNH domain has been identified as a "conformational checkpoint" between DNA binding and cleavage, in which the RNA:DNA complementarity is recognized before the HNH domain assumes an activated conformation.(12) Sm FRET experiments have shown that the presence of DNA mismatc...

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“…Importantly, DNA cleavage efficiency scales with sampling of the HNH domain conformation; a correct conformation enhances the cleavage activity of the RuvC domain 27 . This allostery between HNH and RuvC is believed to result from the hinge comprising two helices that connect RuvC-III to HNH 21,[27][28][29][30][31][32] . Mutations that disrupt one of the two hinge helices lowered the DNA cleavage activity of SpyCas9 27 .…”

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confidence: 99%

Catalytically Enhanced Cas9 through Directed Protein Evolution

Hand

Roth

Smith

et al. 2020

Preprint

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AbstractThe Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 system has found widespread applications in genome manipulations due to its simplicity and effectiveness. Significant efforts in enzyme engineering have been made to improve the CRISPR-Cas9 systems beyond their natural power with additional functionalities such as DNA modification, transcriptional regulation, and high target selectivity1–10. Relatively less attention, however, has been paid to improving the catalytic efficiency of CRISPR-Cas9. Increased catalytic efficiency may be desired in applications where the currently available CRISPR-Cas9 tools are either ineffective4, 11–14 or of low efficiency such as with type II-C Cas915–18 or in non-mammals19, 20. We describe a directed protein evolution method that enables selection of catalytically enhanced CRISPR-Cas9 variants (CECas9). We demonstrate the effectiveness of this method with a previously characterized Type IIC Cas9 from Acidothermus cellulolyticus (AceCas9) with up to 4-fold improvement of in vitro catalytic efficiency, as well as the widely used Streptococcus pyogenes Cas9 (SpyCas9), which showed a 2-fold increase in homology directed repair (HDR)-based gene insertion in human colon cancer cells.

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Key role of the REC lobe during CRISPR–Cas9 activation by ‘sensing’, ‘regulating’, and ‘locking’ the catalytic HNH domain

Cited by 93 publications

References 64 publications

Molecular Dynamics Reveals a DNA-Induced Dynamic Switch Triggering Activation of CRISPR-Cas12a

Molecular Dynamics Reveals a DNA-Induced Dynamic Switch Triggering Activation of CRISPR-Cas12a

Molecular mechanism of off-target effects in CRISPR-Cas9

Catalytically Enhanced Cas9 through Directed Protein Evolution

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