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Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice.

Published on Dec 1, 2015in Genome Biology 13.21
· DOI :10.1186/s13059-015-0653-x
Tomomi Aida11
Estimated H-index: 11
(Tokyo Medical and Dental University),
Keiho Chiyo1
Estimated H-index: 1
(Tokyo Medical and Dental University)
+ 7 AuthorsKohichi Tanaka51
Estimated H-index: 51
(Tokyo Medical and Dental University)
Abstract
Although the CRISPR/Cas system has enabled one-step generation of knockout mice, low success rates of cassette knock-in limit its application range. Here we show that cloning-free, direct nuclear delivery of Cas9 protein complex with chemically synthesized dual RNAs enables highly efficient target digestion, leading to generation of knock-in mice carrying a functional cassette with up to 50% efficiency, compared with just 10% by a commonly used method consisting of Cas9 mRNA and single guide RNA. Our cloning-free CRISPR/Cas system facilitates rapid one-step generation of cassette knock-in mice, accelerating functional genomic research by providing various in vivo genetic tools.
  • References (48)
  • Citations (122)
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References48
Newest
Published on Jun 1, 2015in Neuropsychopharmacology 6.54
Tomomi Aida11
Estimated H-index: 11
(Tokyo Medical and Dental University),
Junichi Yoshida1
Estimated H-index: 1
(Tokyo Medical and Dental University)
+ 16 AuthorsHidenori Aizawa20
Estimated H-index: 20
(Tokyo Medical and Dental University)
Astroglial Glutamate Transporter Deficiency Increases Synaptic Excitability and Leads to Pathological Repetitive Behaviors in Mice
45 Citations Source Cite
Published on Feb 1, 2015in Nature Biotechnology 35.72
Richard L. Frock13
Estimated H-index: 13
,
Jiazhi Hu11
Estimated H-index: 11
+ 3 AuthorsFrederick W. Alt155
Estimated H-index: 155
(Harvard University)
An unbiased genome-wide method reveals on- and off-target DNA cleavage by TALEN and Cas9 nucleases by detecting chromosome translocation events.
297 Citations Source Cite
Published on Feb 1, 2015in Nature Biotechnology 35.72
Xiaoling Wang3
Estimated H-index: 3
,
Yebo Wang5
Estimated H-index: 5
+ 7 AuthorsJiing-Kuan Yee13
Estimated H-index: 13
Off-target cleavage by CAS9 or TALEN genome editing tools is detected by integrase-defective lentiviral vectors.
225 Citations Source Cite
Published on Feb 1, 2015in Nature Biotechnology 35.72
Shengdar Q. Tsai24
Estimated H-index: 24
(Harvard University),
Zongli Zheng24
Estimated H-index: 24
(Karolinska Institutet)
+ 9 AuthorsLong P. Le19
Estimated H-index: 19
(Harvard University)
An unbiased approach for the genome-wide detection of off-target cleavage by CRISPR-Cas9 RNA–guided nucleases reveals wide variability in the off-target activity of different guide RNAs.
662 Citations Source Cite
Published on Jan 1, 2015in Genetics 4.08
Priti Singh2
Estimated H-index: 2
(Cornell University),
John C. Schimenti45
Estimated H-index: 45
(Cornell University),
Ewelina Bolcun-Filas11
Estimated H-index: 11
(Cornell University)
CRISPR/Cas9 system of RNA-guided genome editing is revolutionizing genetics research in a wide spectrum of organisms. Even for the laboratory mouse, a model that has thrived under the benefits of embryonic stem (ES) cell knockout capabilities for nearly three decades, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 technology enables one to manipulate the genome with unprecedented simplicity and speed. It allows generation of null, conditional, precisely mutated, reporter...
160 Citations Source Cite
Published on Jan 1, 2015in Nature Biotechnology 35.72
John A. Zuris15
Estimated H-index: 15
,
David B. Thompson10
Estimated H-index: 10
+ 7 AuthorsDavid R. Liu69
Estimated H-index: 69
(Harvard University)
Efficient protein delivery using cationic lipid transfection reagents enables high efficiency protein-based genome editing in vivo and in vitro.
463 Citations Source Cite
Published on Jan 1, 2015in Methods of Molecular Biology
Le Cong15
Estimated H-index: 15
(Massachusetts Institute of Technology),
Feng Zhang104
Estimated H-index: 104
(Massachusetts Institute of Technology)
Abstract The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is an adaptive immune system that exists in a variety of microbes. It could be engineered to function in eukaryotic cells as a fast, low-cost, efficient, and scalable tool for manipulating genomic sequences. In this chapter, detailed protocols are described for harnessing the CRISPR-Cas9 system from Streptococcus pyogenes to enable RNA-guided genome engineering applications in mammalian cells. We present ...
167 Citations Source Cite
Published on Dec 1, 2014in Nature Communications 12.35
Shota Nakade6
Estimated H-index: 6
,
Takuya Tsubota10
Estimated H-index: 10
+ 8 AuthorsTetsushi Sakuma31
Estimated H-index: 31
One challenge facing the use of programmable nucleases in genome engineering is the requirement for homologous recombination. Here, Nakade et al. harness microhomology-mediated end-joining as a means of inserting exogenous coding sequences into the genome using both TALEN and CRISPR/Cas9 technologies.
188 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
(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 Nov 1, 2014in Nature 41.58
Havva Keskin6
Estimated H-index: 6
,
Ying Shen10
Estimated H-index: 10
+ 5 AuthorsFrancesca Storici19
Estimated H-index: 19
Endogenous RNA transcripts are shown to mediate recombination with yeast chromosomal DNA; as the level of RNAs in the nucleus is quite high, these results may open up new understanding of the plasticity of repair and genome instability mechanisms.
117 Citations Source Cite
Cited By122
Newest
Published on Aug 1, 2019in Current Opinion in Biotechnology 8.38
Hideki Ukai8
Estimated H-index: 8
(University of Tokyo),
Kenta Sumiyama24
Estimated H-index: 24
,
Hiroki R. Ueda43
Estimated H-index: 43
(University of Tokyo)
Systems-biological approaches, such as comprehensive identification and analysis of system components and networks, are necessary to understand design principles of human physiology and pathology. Although reverse genetics using mouse models have been used previously, it is a low throughput method because of the need for repetitive crossing to produce mice having all cells of the body with knock-out or knock-in mutations. Moreover, there are often issues from the interspecific gap between humans...
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Published on Jun 1, 2019in Journal of Autoimmunity 7.61
Nanyang Xiao (Fujian Normal University), Jingjing Wei (Fujian Normal University)+ 6 AuthorsQi Chen (Fujian Normal University)
Abstract TREX1 encodes a major cellular DNA exonuclease. Mutations of this gene in human cause cellular accumulation of DNA that triggers autoimmune diseases including Aicardi–Goutieres Syndrome (AGS) and systemic lupus erythematosus (SLE). We created a lupus mouse model by engineering a D18 N mutation in the Trex1 gene which inactivates the enzyme and has been found in human patients with lupus-like disorders. The Trex1 D18N/D18N mice exhibited systemic inflammation that consistently recapitula...
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Published on Jun 1, 2019in Marine Biotechnology 2.33
Hong Yu16
Estimated H-index: 16
(Ocean University of China),
Huijuan Li1
Estimated H-index: 1
(Ocean University of China)
+ 3 AuthorsShao Jun Du15
Estimated H-index: 15
(University of Maryland, Baltimore)
The Pacific oyster (Crassostrea gigas) is a representative bivalve mollusc that is widely cultured in the world. In recent years, it has become an important model species for ecological, evolutionary, and developmental studies because of its ability to survive in extreme environmental conditions as a sessile filter feeder and its classical mosaic pattern of development. Although the complete genome sequence of C. gigas is available and omics data have been rapidly generated for the past few year...
1 Citations Source Cite
Published on May 1, 2019in Hormones and Behavior 4.42
Kengo Horie3
Estimated H-index: 3
(Laboratory of Molecular Biology),
Kiyoshi Inoue13
Estimated H-index: 13
(Yerkes National Primate Research Center)
+ 6 AuthorsKatsuhiko Nishimori33
Estimated H-index: 33
(Laboratory of Molecular Biology)
Abstract Behavioral neuroendocrinology has benefited tremendously from the use of a wide range of model organisms that are ideally suited for particular questions. However, in recent years the ability to manipulate the genomes of laboratory strains of mice has led to rapid advances in our understanding of the role of specific genes, circuits and neural populations in regulating behavior. While genome manipulation in mice has been a boon for behavioral neuroscience, the intensive focus on the mou...
1 Citations Source Cite
Published on Apr 1, 2019in Journal of Biochemistry 2.35
Kyosuke Kakuda1
Estimated H-index: 1
(Gifu University),
Ayumi Niwa2
Estimated H-index: 2
(Gifu University)
+ 7 AuthorsKazuo Kuwata24
Estimated H-index: 24
(Gifu University)
1 Citations Source Cite
Published on Mar 1, 2019in Analytical Biochemistry 2.27
Bita Ghassemi , Mehdi Shamsara1
Estimated H-index: 1
+ 2 AuthorsMinoo Rassoulzadegan20
Estimated H-index: 20
(University of Nice Sophia Antipolis)
Abstract Animal models possess undeniable utility for progress on biomedical research projects and developmental and disease studies. Transgenic mouse models recreating specific disease phenotypes associated with β-hemoglobinopathies have been developed previously. However, traditional methods for gene targeting in mouse using embryonic stem cells (ESCs) are laborious and time consuming. Recently, CRISPR has been developed to facilitate and improve genomic modifications in mouse or isogenic cell...
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Published on Mar 1, 2019in Vascular Pharmacology 3.61
Joseph M. Miano48
Estimated H-index: 48
(University of Rochester),
Xiaochun Long3
Estimated H-index: 3
(Albany Medical College),
Qing Lyu3
Estimated H-index: 3
(University of Rochester)
Abstract Next generation sequencing has uncovered a trove of short noncoding RNAs (e.g., microRNAs) and long noncoding RNAs (lncRNAs) that act as molecular rheostats in the control of diverse homeostatic processes. Meanwhile, the tsunamic emergence of clustered regularly interspaced short palindromic repeats (CRISPR) editing has transformed our influence over all DNA-carrying entities, heralding global CRISPRization. This is evident in biomedical research where the ease and low-cost of CRISPR ed...
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Published on Mar 1, 2019in Molecular therapy. Nucleic acids 5.66
Lyujie Fang1
Estimated H-index: 1
(Jinan University),
Sandy S. C. Hung12
Estimated H-index: 12
+ 6 AuthorsRaymond C.B. Wong14
Estimated H-index: 14
(University of Melbourne)
Gain-of-function studies often require the tedious cloning of transgene cDNA into vectors for overexpression beyond the physiological expression levels. The rapid development of CRISPR/Cas technology presents promising opportunities to address these issues. Here, we report a simple, cloning-free method to induce gene expression at an endogenous locus using CRISPR/Cas9 activators. Our strategy utilizes synthesized sgRNA expression cassettes to direct a nuclease-null Cas9 complex fused with transc...
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Published on Feb 1, 2019in Alcohol 2.42
Gregg E. Homanics54
Estimated H-index: 54
(University of Pittsburgh)
Abstract Genetically engineered animals are powerful tools that have provided invaluable insights into mechanisms of alcohol action and alcohol-use disorder. Traditionally, production of gene-targeted animals was a tremendously expensive, time consuming, and technically demanding undertaking. However, the recent advent of facile methods for editing the genome at very high efficiency is revolutionizing how these animals are made. While pioneering approaches to create gene-edited animals first use...
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