Cut Site Selection by the Two Nuclease Domains of the Cas9 RNA-guided Endonuclease

Chen, Hongfan; Choi, Jin‐Woo; Bailey, Scott

doi:10.1074/jbc.m113.539726

Cited by 116 publications

(86 citation statements)

References 47 publications

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“…pyogenes Cas9 (hereafter referred to as SpyCas9) is a large (1,368-amino-acid) multidomain and multifunctional DNA endonuclease (Figure 1b). It snips dsDNA 3 bp upstream of the PAM through its two distinct nuclease domains: an HNH-like nuclease domain that cleaves the DNA strand complementary to the guide RNA sequence (target strand), and an RuvC-like nuclease domain responsible for cleaving the DNA strand opposite the complementary strand (nontarget strand) (Figure 2) (13,27,48). In addition to its critical role in CRISPR interference, Cas9 also participates in crRNA maturation and spacer acquisition (32).…”

Section: The Cas9 Enzymementioning

confidence: 99%

CRISPR–Cas9 Structures and Mechanisms

Jiang

Doudna

2017

Annu. Rev. Biophys.

1,560

1,206

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Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes. The use of CRISPR-Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)-CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.

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Section: The Cas9 Enzymementioning

confidence: 99%

CRISPR–Cas9 Structures and Mechanisms

Jiang

Doudna

2017

Annu. Rev. Biophys.

1,560

1,206

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“…Therefore, investigation of such HIV-1 vaccination in various latent reservoir cells and animal models with stable expression of Cas9/LTR-gRNAs presents an important next step to assess the ability of Cas9 to eradicate viral reservoirs in vivo. Moreover, in light of recent data illustrating efficient in vitro genome editing using a mixture of Cas9/gRNA and DNA (39)(40)(41)(42), one may explore various systems for delivery of Cas9/LTR-gRNA via various routes for immunizing high-risk subjects. Once advanced, one may use gene therapies (viral vector and nanoparticle) and transplantation of autologous Cas9/gRNA-modified bone marrow stem/progenitor cells (43,44) or inducible pluripotent stem cells for eradicating HIV-1 infection.…”

Section: Discussionmentioning

confidence: 99%

RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection

Kaminski

Yang

et al. 2014

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

487

475

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Significance For more than three decades since the discovery of HIV-1, AIDS remains a major public health problem affecting greater than 35.3 million people worldwide. Current antiretroviral therapy has failed to eradicate HIV-1, partly due to the persistence of viral reservoirs. RNA-guided HIV-1 genome cleavage by the Cas9 technology has shown promising efficacy in disrupting the HIV-1 genome in latently infected cells, suppressing viral gene expression and replication, and immunizing uninfected cells against HIV-1 infection. These properties may provide a viable path toward a permanent cure for AIDS, and provide a means to vaccinate against other pathogenic viruses. Given the ease and rapidity of Cas9/guide RNA development, personalized therapies for individual patients with HIV-1 variants can be developed instantly.

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“…geewisc.wisc.edu.) 2013; Mali et al 2013a;Ran et al 2013;Chen et al 2014;Cho et al 2014;Fauser et al 2014;Fujii et al 2014;Lin et al 2014;Rong et al 2014;Shen et al 2014). In this approach, pairs of Cas9 nickases are targeted to generate single-strand breaks on opposite strands of the genomic target DNA.…”

Section: The Crispr-cas9 Systemmentioning

confidence: 99%

A CRISPR view of development

et al. 2014

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The CRISPR (clustered regularly interspaced short palindromic repeat)–Cas9 (CRISPR-associated nuclease 9) system is poised to transform developmental biology by providing a simple, efficient method to precisely manipulate the genome of virtually any developing organism. This RNA-guided nuclease (RGN)-based approach already has been effectively used to induce targeted mutations in multiple genes simultaneously, create conditional alleles, and generate endogenously tagged proteins. Illustrating the adaptability of RGNs, the genomes of >20 different plant and animal species as well as multiple cell lines and primary cells have been successfully modified. Here we review the current and potential uses of RGNs to investigate genome function during development.

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Cut Site Selection by the Two Nuclease Domains of the Cas9 RNA-guided Endonuclease

Cited by 116 publications

References 47 publications

CRISPR–Cas9 Structures and Mechanisms

CRISPR–Cas9 Structures and Mechanisms

RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection

A CRISPR view of development

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