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Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA

Published on Mar 1, 2016in Nature Biotechnology 35.72
· DOI :10.1038/nbt.3481
Christopher D. Richardson27
Estimated H-index: 27
Graham Jordan Ray4
Estimated H-index: 4
+ 2 AuthorsJacob E. Corn29
Estimated H-index: 29
The efficiency of homology-directed genome editing with CRISPR-Cas9 is boosted through improved design of donor DNA.
  • References (32)
  • Citations (306)
Published on Nov 13, 2015in Science 41.06
Spencer Knight8
Estimated H-index: 8
(University of California, Berkeley),
Xie L1
Estimated H-index: 1
(University of California, Berkeley)
+ 10 AuthorsMaxime Dahan40
Estimated H-index: 40
(CNRS: Centre national de la recherche scientifique)
The RNA-guided CRISPR-associated protein Cas9 is used for genome editing, transcriptional modulation, and live-cell imaging. Cas9-guide RNA complexes recognize and cleave double-stranded DNA sequences on the basis of 20-nucleotide RNA-DNA complementarity, but the mechanism of target searching in mammalian cells is unknown. Here, we use single-particle tracking to visualize diffusion and chromatin binding of Cas9 in living cells. We show that three-dimensional diffusion dominates Cas9 searching i...
131 Citations Source Cite
Published on Nov 1, 2015in Trends in Biochemical Sciences 15.68
Agnel Sfeir21
Estimated H-index: 21
(NYU: New York University),
Lorraine S. Symington44
Estimated H-index: 44
(CUMC: Columbia University Medical Center)
DNA double-strand breaks (DSBs) disrupt the continuity of chromosomes and their repair by error-free mechanisms is essential to preserve genome integrity. Microhomology-mediated end joining (MMEJ) is an error-prone repair mechanism that involves alignment of microhomologous sequences internal to the broken ends before joining, and is associated with deletions and insertions that mark the original break site, as well as chromosome translocations. Whether MMEJ has a physiological role or is simply...
133 Citations Source Cite
Published on Oct 15, 2015in Annual Review of Microbiology 9.81
Wenyan Jiang11
Estimated H-index: 11
Luciano A. Marraffini33
Estimated H-index: 33
(Rockefeller University)
Prokaryotic CRISPR-Cas loci encode proteins that function as an adaptive immune system against infectious viruses and plasmids. Immunity is mediated by Cas nucleases and small RNA guides, which specify a cleavage site within the genome of the invader. In type II CRISPR-Cas systems, the RNA-guided Cas9 nuclease cleaves the DNA. Cas9 can be reprogrammed to create double-strand DNA breaks in the genomes of a variety of organisms, from bacteria to human cells. Repair of Cas9 lesions by homologous re...
72 Citations Source Cite
Published on May 1, 2015in Nature Biotechnology 35.72
Van Trung Chu12
Estimated H-index: 12
Timm Weber4
Estimated H-index: 4
+ 4 AuthorsRalf Kühn49
Estimated H-index: 49
The efficiency of precise CRISPR/Cas9 genome editing is increased by inhibition of the nonhomologous end joining pathway.
448 Citations Source Cite
Published on Mar 1, 2015in Nature 41.58
Robert Heler4
Estimated H-index: 4
(Rockefeller University),
Poulami Samai8
Estimated H-index: 8
(Rockefeller University)
+ 4 AuthorsLuciano A. Marraffini33
Estimated H-index: 33
(Rockefeller University)
Bacterial CRISPR–Cas loci acquire short phage sequences called spacers that integrate between DNA repeats and how these viral sequences are chosen was unknown; in these studies of the type II CRISPR–Cas system of Streptococcus pyogenes, the Cas9 nuclease known to inactivate invading viral DNA was found to be required for the selection of functional spacers during CRISPR immunity.
144 Citations Source Cite
Published on Dec 15, 2014in eLife 7.62
Steven Lin24
Estimated H-index: 24
(University of California, Berkeley),
Brett T. Staahl13
Estimated H-index: 13
(University of California, Berkeley)
+ 1 AuthorsJennifer A. Doudna92
Estimated H-index: 92
The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (Jinek et al., 2013), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-...
397 Citations Source Cite
Published on Nov 28, 2014in Science 41.06
Jennifer A. Doudna92
Estimated H-index: 92
(University of California, Berkeley),
Emmanuelle Charpentier28
Estimated H-index: 28
(MHH: Hannover Medical School)
The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of...
1,804 Citations Source Cite
Published on Sep 1, 2014in Nature 41.58
Carolin Anders8
Estimated H-index: 8
Ole Niewoehner5
Estimated H-index: 5
+ 1 AuthorsMartin Jinek26
Estimated H-index: 26
Crystal structure of the RNA-guided endonuclease Cas9 bound to a guide RNA and a target DNA duplex reveals how base-specific recognition of a short motif known as PAM in the DNA target results in localized strand separation in the DNA immediately upstream of the PAM, allowing the target DNA strand to hybridize to the guide RNA.
426 Citations Source Cite
Published on Jun 1, 2014in Nature Biotechnology 35.72
Shengdar Q. Tsai24
Estimated H-index: 24
Nicolas Wyvekens3
Estimated H-index: 3
+ 6 AuthorsJ. Keith Joung60
Estimated H-index: 60
(Harvard University)
Monomeric CRISPR-Cas9 nucleases are widely used for targeted genome editing but can induce unwanted off-target mutations with high frequencies. Here we describe dimeric RNA-guided FokI Nucleases (RFNs) that recognize extended sequences and can edit endogenous genes with high efficiencies in human cells. The cleavage activity of an RFN depends strictly on the binding of two guide RNAs (gRNAs) to DNA with a defined spacing and orientation and therefore show improved specificities relative to wild-...
555 Citations Source Cite
Published on Jun 1, 2014in Nature 41.58
Pietro Genovese11
Estimated H-index: 11
(UniSR: Vita-Salute San Raffaele University),
Giulia Schiroli5
Estimated H-index: 5
(UniSR: Vita-Salute San Raffaele University)
+ 13 AuthorsMichael C. Holmes45
Estimated H-index: 45
(Sangamo BioSciences)
Targeted genome editing by artificial nucleases has brought the goal of site-specific transgene integration and gene correction within the reach of gene therapy. However, its application to long-term repopulating haematopoietic stem cells (HSCs) has remained elusive. Here we show that poor permissiveness to gene transfer and limited proficiency of the homology-directed DNA repair pathway constrain gene targeting in human HSCs. By tailoring delivery platforms and culture conditions we overcame th...
278 Citations Source Cite
Cited By306
Published on Mar 8, 2019in Nature Communications 12.35
Grégoire Cullot (French Institute of Health and Medical Research), Julian Boutin (French Institute of Health and Medical Research)+ 17 AuthorsVéronique Guyonnet-Duperat12
Estimated H-index: 12
(French Institute of Health and Medical Research)
CRISPR-Cas9 is a promising technology for genome editing. Here we use Cas9 nuclease-induced double-strand break DNA (DSB) at the UROS locus to model and correct congenital erythropoietic porphyria. We demonstrate that homology-directed repair is rare compared with NHEJ pathway leading to on-target indels and causing unwanted dysfunctional protein. Moreover, we describe unexpected chromosomal truncations resulting from only one Cas9 nuclease-induced DSB in cell lines and primary cells by a p53-de...
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Published on Apr 16, 2019in Scientific Reports 4.12
Tyson R. Shepherd7
Estimated H-index: 7
(MIT: Massachusetts Institute of Technology),
Rebecca R. Du (MIT: Massachusetts Institute of Technology)+ 2 AuthorsMark Bathe26
Estimated H-index: 26
(MIT: Massachusetts Institute of Technology)
Scalable production of kilobase single-stranded DNA (ssDNA) with sequence control has applications in therapeutics, gene synthesis and sequencing, scaffolded DNA origami, and archival DNA memory storage. Biological production of circular ssDNA (cssDNA) using M13 addresses these needs at low cost. However, one unmet goal is to minimize the essential protein coding regions of the exported DNA while maintaining its infectivity and production purity to produce sequences less than 3,000 nt in length,...
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Published on Jan 25, 2019in Scientific Reports 4.12
Henri J. van de Vrugt1
Estimated H-index: 1
(UvA: University of Amsterdam),
Tim Harmsen2
Estimated H-index: 2
(NKI-AVL: Netherlands Cancer Institute)
+ 8 AuthorsRob M.F. Wolthuis (UvA: University of Amsterdam)
Fanconi anemia (FA) is a cancer predisposition syndrome characterized by congenital abnormalities, bone marrow failure, and hypersensitivity to aldehydes and crosslinking agents. For FA patients, gene editing holds promise for therapeutic applications aimed at functionally restoring mutated genes in hematopoietic stem cells. However, intrinsic FA DNA repair defects may obstruct gene editing feasibility. Here, we report on the CRISPR/Cas9-mediated correction of a disruptive mutation in Fancf. Our...
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Published on Mar 18, 2019in Scientific Reports 4.12
Sachiko Okamoto9
Estimated H-index: 9
Yasunori Amaishi3
Estimated H-index: 3
+ 2 AuthorsJunichi Mineno16
Estimated H-index: 16
Target-specific genome editing using engineered nucleases has become widespread in various fields. Long gene knock-in and single-base substitutions can be performed by homologous recombination (HR), but the efficiency is usually very low. To improve the efficiency of knock-in with single-stranded oligo DNA nucleotides (ssODNs), we have investigated optimal design of ssODNs in terms of the blocking mutation, orientation, size, and length of homology arms to explore the optimal parameters of ssODN...
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Published on Mar 25, 2019in Microbial Cell Factories 3.83
Jiao Zhang (BIT: Beijing Institute of Technology), Fayu Yang (BIT: Beijing Institute of Technology)+ 2 AuthorsYi-Xin Huo (BIT: Beijing Institute of Technology)
Background Corynebacterium glutamicum is an important industrial strain for the production of a diverse range of chemicals. Cpf1 nucleases are highly specific and programmable, with efficiencies comparable to those of Cas9. Although the Francisella novicida (Fn) CRISPR-Cpf1 system has been adapted for genome editing in C. glutamicum, the editing efficiency is currently less than 15%, due to false positives caused by the poor targeting efficiency of the crRNA.
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Published on May 25, 2019in Nucleic Acids Research 11.56
Carla M. Mann2
Estimated H-index: 2
(Iowa State University),
Gabriel Martínez-Gálvez4
Estimated H-index: 4
(Mayo Clinic)
+ 8 AuthorsStephen C. Ekker52
Estimated H-index: 52
(Mayo Clinic)
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