Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations

Zuo, Zhicheng; Liu, Jin

doi:10.1038/srep37584

Cited by 120 publications

(160 citation statements)

References 44 publications

(144 reference statements)

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“…S12), allowing the entrance of water molecules, as in a "one-metal aided" architecture (6,26). Concurrently, the nt-DNA strand approaches the RuvC active site with formation of a "two-metal aided" motif (6,27), in which the binding of divalent cations facilitate the movement of the nt-DNA toward the active site (10,28,29). However, limitations of the currently available force fields for Mg ions (30) call for structural validation, which could unambiguously clarify the role of metal ions in the catalysis.…”

Section: Resultsmentioning

confidence: 99%

CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations

Palermo

Miao

Walker

et al. 2017

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

147

251

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CRISPR-Cas9 has become a facile genome editing technology, yet the structural and mechanistic features underlying its function are unclear. Here, we perform extensive molecular simulations in an enhanced sampling regime, using a Gaussian-accelerated molecular dynamics (GaMD) methodology, which probes displacements over hundreds of microseconds to milliseconds, to reveal the conformational dynamics of the endonuclease Cas9 during its activation toward catalysis. We disclose the conformational transition of Cas9 from its apo form to the RNA-bound form, suggesting a mechanism for RNA recruitment in which the domain relocations cause the formation of a positively charged cavity for nucleic acid binding. GaMD also reveals the conformation of a catalytically competent Cas9, which is prone for catalysis and whose experimental characterization is still limited. We show that, upon DNA binding, the conformational dynamics of the HNH domain triggers the formation of the active state, explaining how the HNH domain exerts a conformational control domain over DNA cleavage [Sternberg SH et al. (2015) Nature, 527, 110-113]. These results provide atomic-level information on the molecular mechanism of CRISPR-Cas9 that will inspire future experimental investigations aimed at fully clarifying the biophysics of this unique genome editing machinery and at developing new tools for nucleic acid manipulation based on CRISPR-Cas9.protein-nucleic acid interactions | gene regulation | RNA dynamics | enhanced sampling | free energy L ife sciences are undergoing a transformative phase due to an emerging genome editing technology based on the RNAprogrammable CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) system (1-3). Although this facile genome editing technology is revolutionizing the fields of medicine, pharmaceutics, and even agriculture with the development of drought-resistant crops, the structural and mechanistic details underlying the CRISPR-Cas9 function remain unclear. The CRISPR-Cas9 function begins with the association of Cas9 with a guide RNA, composed of a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA), which enables the recognition and cleavage of matching sequences in double-stranded DNA (3, 4). Upon site-specific recognition of a Protospacer Adjacent Motif (PAM) within the DNA, the latter binds Cas9 matching one strand with the RNA guide (the target DNA strand, t-DNA), whereas the other strand (nontarget DNA, nt-DNA) is displaced. Subsequently, two nuclease domains-HNH and RuvC-perform site-specific cleavages of the t-DNA and nt-DNA strands, respectively.Structural studies have revealed that Cas9 is a large multidomain protein, composed of a recognition lobe, which mediates the nucleic acid binding through three regions (RECI-III), and a nuclease lobe including the RuvC and HNH catalytic cores (Fig. 1) (5). An arginine rich helix (R-rich) bridges the two lobes, whereas the protein C-terminal (Cterm) and PAM-interacting (PI) domains take part in the DNA bindi...

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Section: Resultsmentioning

confidence: 99%

CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations

Palermo

Miao

Walker

et al. 2017

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

147

251

View full text Add to dashboard Cite

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“…The occasional insertion of SNPs 5' of the PAM when a spacer strand repair template was used can be explained by the 3' exonucleolytic activity for the Cas9 nickase RuvC (see model in Figure S1) (Jinek et al 2012;Jiang et al 2015;Zuo and Liu 2016;Stephenson et al 2018). This nickase resects single-stranded DNA in vitro up to 10 nucleotides in the 3' -5' direction on the strand it nicks (Stephenson et al 2018).…”

Section: Precise Repair From Homologous Single-stranded Oligonucleotimentioning

confidence: 99%

Strategies for Efficient Genome Editing Using CRISPR-Cas9

Farboud

Severson

Meyer

2018

Preprint

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The targetable DNA endonuclease CRISPR-Cas9 has transformed analysis of biological processes by enabling robust genome editing in model and non-model organisms. Although rules directing Cas9 to its target DNA via a guide RNA are straightforward, wide variation occurs in editing efficiency and repair outcomes, both imprecise error-prone repair and precise templated repair. We found that imprecise and precise DNA repair from double-stranded breaks (DSBs) is asymmetric, favoring repair in one direction. Using this knowledge, we designed RNA guides and repair templates that increased the frequency of imprecise insertions and deletions and greatly enhanced precise insertion of point mutations in Caenorhabditis elegans. We devised strategies to insert long (10 kb) exogenous sequences or incorporate multiple nucleotide substitutions at considerable distance from DSBs. We expanded the repertoire of co-conversion markers appropriate for diverse nematode species. These selectable markers enable rapid identification of Cas9-edited animals also likely to carry edits in desired targets. Lastly, we explored the timing, location, frequency, sex-dependence, and categories of DSB repair events by developing loci with allele-specific Cas9 targets that can be contributed during mating from either male or hermaphrodite germ cells. Our studies revealed a striking difference in editing efficiency between maternally and paternally contributed genomes. Furthermore, imprecise repair and precise repair from exogenous repair templates occur with high frequency before and after fertilization. Our strategies enhance Cas9 targeting efficiency, lend insight into the timing and mechanisms of DSB repair, and establish guidelines for achieving predictable precise and imprecise repair outcomes with high frequency.

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“…Thus, the transition of the HNH domain that leads Cas9 to adopt conformations beyond those solved by the crystal structures should be crucial for the DNA cleavage. In addition, a previous single‐molecule study implied the conformational flexibility during the DNA binding process (Singh et al , ), and molecular dynamics simulations have also shed light on the importance of the flexible movements of the Cas9 domains in the sgRNA/DNA binding (Palermo et al , ; Zuo & Liu, ; Zheng, ). Thus, the flexibility of the Cas9 domain configuration could be an important factor in the Cas9 catalytic processes.…”

Section: Introductionmentioning

confidence: 99%

Real‐time observation of flexible domain movements in CRISPR–Cas9

et al. 2018

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The CRISPR‐associated protein Cas9 is widely used for genome editing because it cleaves target DNA through the assistance of a single‐guide RNA (sgRNA). Structural studies have revealed the multi‐domain architecture of Cas9 and suggested sequential domain movements of Cas9 upon binding to the sgRNA and the target DNA. These studies also hinted at the flexibility between domains; however, it remains unclear whether these flexible movements occur in solution. Here, we directly observed dynamic fluctuations of multiple Cas9 domains, using single‐molecule FRET. We found that the flexible domain movements allow Cas9 to adopt transient conformations beyond those captured in the crystal structures. Importantly, the HNH nuclease domain only accessed the DNA cleavage position during such flexible movements, suggesting the importance of this flexibility in the DNA cleavage process. Our FRET data also revealed the conformational flexibility of apo‐Cas9, which may play a role in the assembly with the sgRNA. Collectively, our results highlight the potential role of domain fluctuations in driving Cas9‐catalyzed DNA cleavage.

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Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations

Cited by 120 publications

References 44 publications

CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations

CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations

Strategies for Efficient Genome Editing Using CRISPR-Cas9

Real‐time observation of flexible domain movements in CRISPR–Cas9

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