CRISPR–Cas9 Structures and Mechanisms

Jiang, Fuguo; Doudna, Jennifer A.

doi:10.1146/annurev-biophys-062215-010822

Cited by 1,545 publications

(1,304 citation statements)

References 113 publications

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“…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 binding process.…”

mentioning

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.

151

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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“…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 binding process.…”

mentioning

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.

151

251

View full text Add to dashboard Cite

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“…There is considerable diversity among CRISPR/Cas systems [reviewed in (66)], but the widely used Streptococcus pyogenes ( Spy ) Type II CRISPR/Cas9 highlights key features of mechanism (67,68). CRISPR/Cas9 is an RNP, composed of RNA and polypeptide components.…”

Section: Discussionmentioning

confidence: 99%

Initiation of homologous recombination at DNA nicks

Maizels

Davis

2018

Nucleic Acids Research

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Discontinuities in only a single strand of the DNA duplex occur frequently, as a result of DNA damage or as intermediates in essential nuclear processes and DNA repair. Nicks are the simplest of these lesions: they carry clean ends bearing 3′-hydroxyl groups that can undergo ligation or prime new DNA synthesis. In contrast, single-strand breaks also interrupt only one DNA strand, but they carry damaged ends that require clean-up before subsequent steps in repair. Despite their apparent simplicity, nicks can have significant consequences for genome stability. The availability of enzymes that can introduce a nick almost anywhere in a large genome now makes it possible to systematically analyze repair of nicks. Recent experiments demonstrate that nicks can initiate recombination via pathways distinct from those active at double-strand breaks (DSBs). Recombination at targeted DNA nicks can be very efficient, and because nicks are intrinsically less mutagenic than DSBs, nick-initiated gene correction is useful for genome engineering and gene therapy. This review revisits some physiological examples of recombination at nicks, and outlines experiments that have demonstrated that nicks initiate homology-directed repair by distinctive pathways, emphasizing research that has contributed to our current mechanistic understanding of recombination at nicks in mammalian cells.

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“…35 NHEJ, as a primary and efficient repair mechanism, repairs lesions by connecting the two ends of the DSBs, and frequently gives rise to small insertions or deletions (indels) at the breaking site without the existence of a DNA template. Target genes can be disrupted via these indels-mediated reading frame changes, causing mRNA degradation or non-functional protein production 36.…”

Section: Origin Development and Mechanism Of Crispr-cas9 Systemmentioning

confidence: 99%

Applications and advances of CRISPR-Cas9 in cancer immunotherapy

Xia

Wang

et al. 2018

J Med Genet

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Immunotherapy has emerged as one of the most promising therapeutic strategies in cancer. The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (CRISPR-Cas9) system, as an RNA-guided genome editing technology, is triggering a revolutionary change in cancer immunotherapy. With its versatility and ease of use, CRISPR-Cas9 can be implemented to fuel the production of therapeutic immune cells, such as construction of chimeric antigen receptor T (CAR-T) cells and programmed cell death protein 1 knockout. Therefore, CRISPR-Cas9 technology holds great promise in cancer immunotherapy. In this review, we will introduce the origin, development and mechanism of CRISPR-Cas9. Also, we will focus on its various applications in cancer immunotherapy, especially CAR-T cell-based immunotherapy, and discuss the potential challenges it faces.

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CRISPR–Cas9 Structures and Mechanisms

Cited by 1,545 publications

References 113 publications

CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations

CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations

Initiation of homologous recombination at DNA nicks

Applications and advances of CRISPR-Cas9 in cancer immunotherapy

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