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Nucleosomes inhibit target cleavage by CRISPR-Cas9 in vivo

Published on Sep 18, 2018in Proceedings of the National Academy of Sciences of the United States of America9.58
· DOI :10.1073/pnas.1810062115
Robert M. Yarrington7
Estimated H-index: 7
(UofU: University of Utah),
Surbhi Verma6
Estimated H-index: 6
(UofU: University of Utah)
+ 2 AuthorsDana Carroll43
Estimated H-index: 43
(UofU: University of Utah)
Abstract
Genome editing with CRISPR-Cas nucleases has been applied successfully to a wide range of cells and organisms. There is, however, considerable variation in the efficiency of cleavage and outcomes at different genomic targets, even within the same cell type. Some of this variability is likely due to the inherent quality of the interaction between the guide RNA and the target sequence, but some may also reflect the relative accessibility of the target. We investigated the influence of chromatin structure, particularly the presence or absence of nucleosomes, on cleavage by the Streptococcus pyogenes Cas9 protein. At multiple target sequences in two promoters in the yeast genome, we find that Cas9 cleavage is strongly inhibited when the DNA target is within a nucleosome. This inhibition is relieved when nucleosomes are depleted. Remarkably, the same is not true of zinc-finger nucleases (ZFNs), which cleave equally well at nucleosome-occupied and nucleosome-depleted sites. These results have implications for the choice of specific targets for genome editing, both in research and in clinical and other practical applications.
  • References (52)
  • Citations (27)
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References52
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#1Chao Yan (PSU: Pennsylvania State University)H-Index: 3
#2Hengye Chen (PSU: Pennsylvania State University)H-Index: 1
Last. Lu Bai (PSU: Pennsylvania State University)H-Index: 15
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Summary Nucleosomes present a barrier for the binding of most transcription factors (TFs). However, special TFs known as nucleosome-displacing factors (NDFs) can access embedded sites and cause the depletion of the local nucleosomes as well as repositioning of the neighboring nucleosomes. Here, we developed a novel high-throughput method in yeast to identify NDFs among 104 TFs and systematically characterized the impact of orientation, affinity, location, and copy number of their binding motifs ...
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#1Nicole M. GaudelliH-Index: 6
#2Alexis C. KomorH-Index: 13
Last. David R. LiuH-Index: 73
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A new DNA ‘base editor’ can change targeted A•T base pairs to G•C, allowing disease-associated mutations to be corrected and disease-suppressing mutations to be introduced into cells.
457 CitationsSource
#1Xiaoyu Chen (LEI: Leiden University)H-Index: 7
#2Jin Liu (LEI: Leiden University)H-Index: 9
Last. Manuel A. F. V. Gonçalves (LEI: Leiden University)H-Index: 23
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#1Kristopher Torp Jensen (University of Cambridge)H-Index: 3
#2Lasse Fløe (AU: Aarhus University)H-Index: 2
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CRISPR-Cas9 systems have emerged as the method of choice for genome editing, but large variations in on-target efficiencies continue to limit their applicability. Here, we investigate the effect of chromatin accessibility on Cas9-mediated gene editing efficiency for 20 gRNAs targeting 10 genomic loci in HEK293T cells using both SpCas9 and the eSpCas9(1.1) variant. Our study indicates that gene editing is more efficient in euchromatin than in heterochromatin, and we validate this finding in HeLa ...
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#2Jacob E. Corn (University of California, Berkeley)H-Index: 32
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The CRISPR-Cas genome editing system is very powerful. The format of the CRISPR reagents and the means of delivery are often important factors in targeting efficiency. Delivery of recombinant Cas9 protein and guide RNA (gRNA) as a preformed ribonucleoprotein (RNP) complex has recently emerged as a powerful and general approach to genome editing. Here we outline methods to produce and deliver Cas9 RNPs. A donor DNA carrying desired sequence changes can also be included to program precise sequence...
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#2Josh Cutts (ASU: Arizona State University)H-Index: 6
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In order to efficiently edit eukaryotic genomes, it is critical to test the impact of chromatin dynamics on CRISPR/Cas9 function and develop strategies to adapt the system to eukaryotic contexts. So far, research has extensively characterized the relationship between the CRISPR endonuclease Cas9 and the composition of the RNA–DNA duplex that mediates the system’s precision. Evidence suggests that chromatin modifications and DNA packaging can block eukaryotic genome editing by custom-built DNA en...
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#1Alexis C. Komor (Broad Institute)H-Index: 13
#2Ahmed H. Badran (Broad Institute)H-Index: 13
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The CRISPR-Cas9 RNA-guided DNA endonuclease has contributed to an explosion of advances in the life sciences that have grown from the ability to edit genomes within living cells. In this Review, we summarize CRISPR-based technologies that enable mammalian genome editing and their various applications. We describe recent developments that extend the generality, DNA specificity, product selectivity, and fundamental capabilities of natural CRISPR systems, and we highlight some of the remarkable adv...
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Abstract Nucleosomes affect Cas9 binding and activity at on-target sites, but their impact at off-target sites is unknown. To investigate how nucleosomes affect Cas9 cleavage at off-target sites in vitro, we used a single guide RNA (sgRNA) that has been previously shown to efficiently direct Cas9 cleavage at the edge of the strongly positioned 601 nucleosome. Our data indicate that single mismatches between the sgRNA and DNA target have relatively little effect on Cas9 cleavage of naked DNA subs...
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#1Mark A. DeWitt (University of California, Berkeley)H-Index: 10
#2Wendy Magis (Children's Hospital Oakland Research Institute)H-Index: 4
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Sickle cell disease is a genetic disorder caused by a mutation in one of the hemoglobin genes, which causes deformation of red blood cells and results in occlusion of blood vessels, severe pain crises, and progressive organ injury. To correct the mutation that causes this disease, DeWitt et al . modified hematopoietic stem cells from sickle cell disease patients using a CRISPR/Cas9 gene editing approach. The authors showed that the corrected cells successfully engrafted in a mouse model and prod...
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INTRODUCTION To combat invading pathogens, cells develop an adaptive immune response by changing their own genetic information. In vertebrates, the generation of genetic variation (somatic hypermutation) is an essential process for diversification and affinity maturation of antibodies that function to detect and sequester various foreign biomolecules. The activation-induced cytidine deaminase (AID) carries out hypermutation by modifying deoxycytidine bases in the variable region of the immunoglo...
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Abstract Base editing is a form of genome editing that can directly convert a single base (C or A) to another base (T or G), which is of great potential in biomedical applications. The broad application of base editing is limited by its low activity and specificity which still needs to be resolved. To address this, a simple and quick method for the determination of its activity/specificity is highly desired. Here, we developed a novel system, which could be harnessed for quick detection of editi...
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Cas9 is an RNA-guided endonuclease in the bacterial CRISPR-Cas immune system and a popular tool for genome editing. The most commonly used Cas9 variant, Streptococcus pyogenes Cas9 (SpCas9), is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific than wild-type (WT) SpCas9. However, systematic comparisons of the cleavage activities of these Cas9 variants have not been reported. In this study,...
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