CRISPR Cas9 searches for a protospacer adjacent motif by one-dimensional diffusion

Globyte, Viktorija; Lee, Seung Hwan; Bae, Taegun; Joo, Chirlmin

doi:10.1101/264879

Cited by 6 publications

(3 citation statements)

References 38 publications

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“…Neutralizing mutations in these positive patches reduce the lifetimes of diffusing Cascade complexes on non-specific DNA and decrease the in vivo interference efficiency. Facilitated diffusion is likely a conserved search mechanism among all CRISPR systems (Globyte et al, 2018; Xue et al, 2017). Cascade target recognition and stable R-loop locking proceeds via at least two temporally distinct intermediates.…”

Section: Discussionmentioning

confidence: 99%

Assembly and Translocation of a CRISPR-Cas Primed Acquisition Complex

Dillard

Brown

Johnson

et al. 2018

Cell

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CRISPR-Cas systems confer an adaptive immunity against viruses. Following viral injection, Cas1-Cas2 integrates segments of the viral genome (spacers) into the CRISPR locus. In addition, efficient “primed” spacer acqui sition and viral degradation (interference) both require the Cascade complex along with the Cas3 helicase/nuclease. Here, we present single-molecule characterization of the Thermobifida fusca (Tfu) primed acquisition complex (PAC). We show that TfuCascade rapidly samples non-specific DNA via facilitated one-dimensional diffusion. Cas3 loads at target-bound Cascade and the Cascade/Cas3 complex translocates via a looped DNA intermediate. Cascade/Cas3 complexes stall at diverse protein roadblocks, resulting in a double strand break at the stall site. In contrast, Cas1-Cas2 samples DNA transiently via 3D collisions. Moreover, Cas1-Cas2 associates with Cascade and translocates with Cascade/Cas3, forming the PAC. PACs can displace different protein roadblocks, suggesting a mechanism for long-range spacer acquisition. This work provides a molecular basis for the coordinated steps in CRISPR-based adaptive immunity.

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Section: Discussionmentioning

confidence: 99%

Assembly and Translocation of a CRISPR-Cas Primed Acquisition Complex

Dillard

Brown

Johnson

et al. 2018

Cell

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“…The Cas/gRNA complex negotiates the DNA strand for an appropriate PAM sequence, after which target recognition occurs if strong complementarity between the gRNA seed region and target DNA is found. The formation of the RNA-DNA hybrid facilitates conformational changes within the Cas protein that triggers the nuclease activity (Globyte et al, 2019;Jiang & Doudna, 2017;Sternberg et al, 2015;Wu et al, 2014). The careful design, evaluation, and selection of gRNAs are therefore essential for a successful experiment.…”

Section: Evaluation Of Editing Reagents Prior To Crop Transformationmentioning

confidence: 99%

Targeted mutagenesis with sequence‐specific nucleases for accelerated improvement of polyploid crops: Progress, challenges, and prospects

2023

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Many of the world's most important crops are polyploid. The presence of more than two sets of chromosomes within their nuclei and frequently aberrant reproductive biology in polyploids present obstacles to conventional breeding. The presence of a larger number of homoeologous copies of each gene makes random mutation breeding a daunting task for polyploids. Genome editing has revolutionized improvement of polyploid crops as multiple gene copies and/or alleles can be edited simultaneously while preserving the key attributes of elite cultivars. Most genome‐editing platforms employ sequence‐specific nucleases (SSNs) to generate DNA double‐stranded breaks at their target gene. Such DNA breaks are typically repaired via the error‐prone nonhomologous end‐joining process, which often leads to frame shift mutations, causing loss of gene function. Genome editing has enhanced the disease resistance, yield components, and end‐use quality of polyploid crops. However, identification of candidate targets, genotyping, and requirement of high mutagenesis efficiency remain bottlenecks for targeted mutagenesis in polyploids. In this review, we will survey the tremendous progress of SSN‐mediated targeted mutagenesis in polyploid crop improvement, discuss its challenges, and identify optimizations needed to sustain further progress.

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“…Numerous experimental studies point to a model for target search, in which PAM recognition acts as an obligate first step during protospacer recognition and leads directly to local DNA unwinding. [57][58][59][60] The search is largely threedimensional, 57,61,62 and sampling times at off-target sites are a function of the degree of gRNA-DNA complementarity. [63][64][65] Targeting by Cas9-gRNA is most sensitive to mismatches in the PAM-proximal region of the DNA target site, a phenomenon that results at least, in part, from preordering of the gRNA ''seed'' sequence.…”

Section: Crispr-cas Diversity and Classificationmentioning

confidence: 99%

Harnessing “A Billion Years of Experimentation”: The Ongoing Exploration and Exploitation of CRISPR–Cas Immune Systems

Klompe

Sternberg

2018

The CRISPR Journal

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The famed physicist-turned-biologist, Max Delbrü ck, once remarked that, for physicists, ''the field of bacterial viruses is a fine playground for serious children who ask ambitious questions.'' Early discoveries in that playground helped establish molecular genetics, and half a century later, biologists delving into the same field have ushered in the era of precision genome engineering. The focus has of course shifted-from bacterial viruses and their mechanisms of infection to the bacterial hosts and their mechanisms of immunity-but it is the very same evolutionary arms race that continues to awe and inspire researchers worldwide. In this review, we explore the remarkable diversity of CRISPR-Cas adaptive immune systems, describe the molecular components that mediate nucleic acid targeting, and outline the use of these RNA-guided machines for biotechnology applications. CRISPR-Cas research has yielded far more than just Cas9-based genome-editing tools, and the wide-reaching, innovative impacts of this fascinating biological playground are sure to be felt for years to come.

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CRISPR Cas9 searches for a protospacer adjacent motif by one-dimensional diffusion

Cited by 6 publications

References 38 publications

Assembly and Translocation of a CRISPR-Cas Primed Acquisition Complex

Assembly and Translocation of a CRISPR-Cas Primed Acquisition Complex

Targeted mutagenesis with sequence‐specific nucleases for accelerated improvement of polyploid crops: Progress, challenges, and prospects

Harnessing “A Billion Years of Experimentation”: The Ongoing Exploration and Exploitation of CRISPR–Cas Immune Systems

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