Match!

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

Published on Mar 1, 2016in Nature Biotechnology36.558
· DOI :10.1038/NBT.3481
Christopher D. Richardson5
Estimated H-index: 5
(University of California, Berkeley),
Graham J. Ray4
Estimated H-index: 4
(University of California, Berkeley)
+ 2 AuthorsJacob E. Corn34
Estimated H-index: 34
(University of California, Berkeley)
Sources
Abstract
The efficiency of homology-directed genome editing with CRISPR-Cas9 is boosted through improved design of donor DNA.
  • References (33)
  • Citations (477)
📖 Papers frequently viewed together
923 Citations
6 Authors (F. Ann Ran, ..., Feng Zhang)
4,532 Citations
952 Citations
78% of Scinapse members use related papers. After signing in, all features are FREE.
References33
Newest
#1Spencer C. Knight (University of California, Berkeley)H-Index: 5
#1Spencer Knight (University of California, Berkeley)H-Index: 7
Last. Robert TjianH-Index: 110
view all 13 authors...
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...
183 CitationsSource
#1Agnel Sfeir (NYU: New York University)H-Index: 23
#2Lorraine S. Symington (CUMC: Columbia University Medical Center)H-Index: 46
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...
198 CitationsSource
#1Wenyan Jiang (Rockefeller University)H-Index: 13
#2Luciano A. Marraffini (Rockefeller University)H-Index: 38
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...
91 CitationsSource
1 CitationsSource
#1Takeshi Maruyama (MIT: Massachusetts Institute of Technology)H-Index: 14
#2Stephanie K. Dougan (MIT: Massachusetts Institute of Technology)H-Index: 28
Last. Hidde L. Ploegh (MIT: Massachusetts Institute of Technology)H-Index: 117
view all 6 authors...
The efficiency of homologous recombination-based Cas9 genome editing is increased by inhibiting non-homologous end joining.
603 CitationsSource
#1Van Trung Chu (MDC: Max Delbrück Center for Molecular Medicine)H-Index: 12
#2Timm Weber (MDC: Max Delbrück Center for Molecular Medicine)H-Index: 4
Last. Ralf Kühn (MDC: Max Delbrück Center for Molecular Medicine)H-Index: 51
view all 7 authors...
The efficiency of precise CRISPR/Cas9 genome editing is increased by inhibition of the nonhomologous end joining pathway.
605 CitationsSource
#1Robert Heler (Rockefeller University)H-Index: 7
#2Poulami Samai (Rockefeller University)H-Index: 10
Last. Luciano A. Marraffini (Rockefeller University)H-Index: 38
view all 7 authors...
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.
187 CitationsSource
#1Steven Lin (University of California, Berkeley)H-Index: 26
#2Brett T. Staahl (University of California, Berkeley)H-Index: 14
Last. Jennifer A. Doudna (University of California, Berkeley)H-Index: 103
view all 4 authors...
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-...
575 CitationsSource
#1Jennifer A. Doudna (University of California, Berkeley)H-Index: 103
#2Emmanuelle Charpentier (MHH: Hannover Medical School)H-Index: 30
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...
2,447 CitationsSource
#1C. Anders (UZH: University of Zurich)H-Index: 10
#2Ole Niewoehner (UZH: University of Zurich)H-Index: 5
Last. Martin Jinek (UZH: University of Zurich)H-Index: 31
view all 4 authors...
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.
565 CitationsSource
Cited By477
Newest
#1Yuyuan Wang (UW: University of Wisconsin-Madison)H-Index: 9
#2Pawan K Shahi (UW: University of Wisconsin-Madison)H-Index: 5
Last. Shaoqin GongH-Index: 56
view all 10 authors...
Abstract Efficient delivery of hydrophilic drugs, nucleic acids, proteins, and any combination thereof is essential for various biomedical applications. Herein, we report a straightforward, yet versatile approach to efficiently encapsulate and deliver various hydrophilic payloads using a pH-responsive silica–metal–organic framework hybrid nanoparticle (SMOF NP) consisting of both silica and zeolitic imidazole framework (ZIF). This unique SMOF NP offers a high loading content and efficiency, exce...
2 CitationsSource
#1Patrick Pausch (University of California, Berkeley)
#2Basem Al-Shayeb (University of California, Berkeley)H-Index: 7
Last. Jennifer A. DoudnaH-Index: 103
view all 10 authors...
CRISPR-Cas systems are found widely in prokaryotes, where they provide adaptive immunity against virus infection and plasmid transformation. We describe a minimal functional CRISPR-Cas system, comprising a single ~70-kilodalton protein, CasΦ, and a CRISPR array, encoded exclusively in the genomes of huge bacteriophages. CasΦ uses a single active site for both CRISPR RNA (crRNA) processing and crRNA-guided DNA cutting to target foreign nucleic acids. This hypercompact system is active in vitro an...
Source
#1Peter TennantH-Index: 9
Last. Margaret A. KeighrenH-Index: 16
view all 15 authors...
Advances in genome editing technologies have created opportunities to treat rare genetic diseases, which are often overlooked in terms of therapeutic development. Nonetheless, substantial challenges remain: namely, achieving therapeutically beneficial levels and kinds of editing in the right cell type(s). Here we describe the development of FIVER (fluorescent in vivo editing reporter) - a modular toolkit for in vivo detection of genome editing with distinct fluorescent read-outs for non-homologo...
Source
#1Daniel RosenblumH-Index: 7
#2Anna GutkinH-Index: 2
Last. Dan PeerH-Index: 38
view all 4 authors...
Abstract CRISPR/Cas systems (clustered regularly interspaced short palindromic repeats) have emerged as powerful tools to manipulate the genome for both research and therapeutic purposes. However, the clinical use of this system is hindered by multiple challenges, such as the rate of off-target effects, editing efficiency, the efficacy of HDR, immunogenicity, as well as development of efficient and safe delivery vehicles that can carry these compounds. Tremendous efforts are being conducted to o...
Source
#1Jiasong Chang (SWU: Southwest University)H-Index: 7
#2Xiaoxu Chen (SWU: Southwest University)H-Index: 2
Last. Qingyou Xia (SWU: Southwest University)H-Index: 44
view all 9 authors...
Abstract The CRISPR/Cas (clustered regularly interspaced short palindromic repeat technology/CRISPR-associated protein) is a widely used and powerful research tool in biosciences and a promising therapeutic agent for treating genetic diseases. Mutations induced by Cas9 are generally considered stochastic and unpredictable, thus hindering its applications where precise genetic alternations are required. Here, through deep sequencing and analysis of genome editing outcomes of multiple sites in fou...
Source
#1James Kuo (Wyss Institute for Biologically Inspired Engineering)H-Index: 8
#2Ruoshi Yuan (Harvard University)H-Index: 11
Last. Pamela A. Silver (Wyss Institute for Biologically Inspired Engineering)H-Index: 95
view all 5 authors...
In synthetic circuits, CRISPR-Cas systems have been used effectively for endpoint changes from an initial state to a final state, such as in logic gates. Here, we use deactivated Cas9 (dCas9) and deactivated Cas12a (dCas12a) to construct dynamic RNA ring oscillators that cycle continuously between states over time in bacterial cells. While our dCas9 circuits using 103-nt guide RNAs showed irregular fluctuations with a wide distribution of peak-to-peak period lengths averaging approximately nine ...
Source
#1Yue Gong (Peking Union Medical College)H-Index: 1
#2Yanwei Bi (Peking Union Medical College)H-Index: 3
Last. Shaozhong Dong (Peking Union Medical College)H-Index: 3
view all 11 authors...
Efficient, accurate and convenient foreign-gene insertion strategies are crucial for the high-throughput and rapid construction of large DNA viral vectors, but relatively inefficient and labour-intensive methods have limited the application of recombinant viruses. In this study, we applied the nonhomologous insertion (NHI) strategy, which is based on the nonhomologous end joining (NHEJ) repair pathway. Compared to the currently used homologous recombination (HR) strategy, we obtained a higher ef...
Source
#1Alan S. Wang (University of California, Berkeley)
#2Leo C. Chen (University of California, Berkeley)
Last. Jonathan Vu (University of California, Berkeley)H-Index: 3
view all 15 authors...
Summary Cas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly in vitro but turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about the factors responsible for dislodging Cas9 or how they influence genome engineering. Unbiased detection through proximity labeling of transient protein interactions in cell-free Xenopus laevis egg extract identified the dimeric histone chaperone facilitates chromatin transcription (FACT) as an interacto...
Source
#1Effimia Christidi (UBC: University of British Columbia)H-Index: 2
#2Haojun Huang (UBC: University of British Columbia)H-Index: 1
Last. Liam R. Brunham (UBC: University of British Columbia)H-Index: 27
view all 10 authors...
Doxorubicin is a potent anticancer drug used to treat a variety of cancer types. However, its use is limited by doxorubicin-induced cardiotoxicity (DIC). A missense variant in the RARG gene (S427L; rs2229774) has been implicated in susceptibility to DIC in a genome wide association study. The goal of this study was to investigate the functional role of this RARG variant in DIC. We used induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) from patients treated with doxorubicin. iPSC-CM...
1 CitationsSource
#1Andrew V. Anzalone (Broad Institute)H-Index: 2
#2Luke W. Koblan (Broad Institute)H-Index: 7
Last. David R. Liu (Broad Institute)H-Index: 82
view all 3 authors...
The development of new CRISPR–Cas genome editing tools continues to drive major advances in the life sciences. Four classes of CRISPR–Cas-derived genome editing agents—nucleases, base editors, transposases/recombinases and prime editors—are currently available for modifying genomes in experimental systems. Some of these agents have also moved rapidly into the clinic. Each tool comes with its own capabilities and limitations, and major efforts have broadened their editing capabilities, expanded t...
2 CitationsSource