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Mechanistic Studies of CRISPR Protei...

Knight, Spencer Charles.

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Mechanistic Studies of CRISPR Proteins in Living Cells.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Mechanistic Studies of CRISPR Proteins in Living Cells./
Author:	Knight, Spencer Charles.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2017,
Description:	128 p.
Notes:	Source: Dissertation Abstracts International, Volume: 78-11(E), Section: B.
Contained By:	Dissertation Abstracts International78-11B(E).
Subject:	Chemistry. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=10281499
ISBN:	9780355033731

Mechanistic Studies of CRISPR Proteins in Living Cells.
Knight, Spencer Charles.

Mechanistic Studies of CRISPR Proteins in Living Cells. - Ann Arbor : ProQuest Dissertations & Theses, 2017 - 128 p.

Source: Dissertation Abstracts International, Volume: 78-11(E), Section: B.

Thesis (Ph.D.)--University of California, Berkeley, 2017.

The discovery of the CRISPR-Cas9 protein has enabled facile genome editing in living cells and organisms. Structural and biochemical studies of Cas9 endonucleases have provided critical information about the core molecular requirements of the RNA-guided DNA cleavage reaction, but questions remain about how this bacterial protein navigates chromatin to identify DNA targets within living eukaryotic cells. In particular, the in vivo kinetics of on- versus off-target binding and Cas9 dependence on chromatin environment remain largely unknown. Here we present a single-molecule analysis of Cas9 searching in living mammalian cells. We provide evidence for a diffusion- dominated search mechanism and show that off-target binding at PAMs and short seed sequences is, on average, short-lived (milleseconds to seconds) by both single- molecule and bulk imaging techniques. The differential behavior of Cas9 in closed-off (heterochromatic) versus open (euchromatic) regions of the genome is also explored via heterochromatin protein 1 (HP1) staining and nuclear masking of single-particle trajectories. Comparative analysis of trajectories from these two regions suggests that Cas9 undersamples heterochromatin and moves more compactly within these regions. These data provide the first direct visualization of Cas9 searching in living cells and offer quantitative insights into how Cas9 navigates hierarchical organization of DNA within a eukaryotic nucleus. We additionally present mechanistic investigations of unrelated CRISPR-C2c2 proteins and identify two distinct enzymatic activities-- pre- CRISPR RNA (pre-crRNA) processing and crRNA-stimulated general RNase activity. Collectively, this work expands our mechanistic understanding of CRISPR biology and highlights the utility of these enzymatically diverse proteins to be harnessed for biotechnological applications.

ISBN: 9780355033731Subjects--Topical Terms:

516420
Chemistry.

Mechanistic Studies of CRISPR Proteins in Living Cells.
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520 $a The discovery of the CRISPR-Cas9 protein has enabled facile genome editing in living cells and organisms. Structural and biochemical studies of Cas9 endonucleases have provided critical information about the core molecular requirements of the RNA-guided DNA cleavage reaction, but questions remain about how this bacterial protein navigates chromatin to identify DNA targets within living eukaryotic cells. In particular, the in vivo kinetics of on- versus off-target binding and Cas9 dependence on chromatin environment remain largely unknown. Here we present a single-molecule analysis of Cas9 searching in living mammalian cells. We provide evidence for a diffusion- dominated search mechanism and show that off-target binding at PAMs and short seed sequences is, on average, short-lived (milleseconds to seconds) by both single- molecule and bulk imaging techniques. The differential behavior of Cas9 in closed-off (heterochromatic) versus open (euchromatic) regions of the genome is also explored via heterochromatin protein 1 (HP1) staining and nuclear masking of single-particle trajectories. Comparative analysis of trajectories from these two regions suggests that Cas9 undersamples heterochromatin and moves more compactly within these regions. These data provide the first direct visualization of Cas9 searching in living cells and offer quantitative insights into how Cas9 navigates hierarchical organization of DNA within a eukaryotic nucleus. We additionally present mechanistic investigations of unrelated CRISPR-C2c2 proteins and identify two distinct enzymatic activities-- pre- CRISPR RNA (pre-crRNA) processing and crRNA-stimulated general RNase activity. Collectively, this work expands our mechanistic understanding of CRISPR biology and highlights the utility of these enzymatically diverse proteins to be harnessed for biotechnological applications.
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