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 Tanaka49
Estimated H-index: 49
(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 (115)
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References48
Published on Jan 1, 2015in Methods of Molecular Biology
Le Cong15
Estimated H-index: 15
(Massachusetts Institute of Technology),
Feng Zhang101
Estimated H-index: 101
(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 ...
153 Citations Source Cite
Published on Mar 1, 2014in Cell Research 15.39
Tang Hai13
Estimated H-index: 13
,
Fei Teng7
Estimated H-index: 7
+ 2 AuthorsQi Zhou41
Estimated H-index: 41
The pig is an important livestock for food supply and an ideal model for various human diseases. Efficient and precise genetic engineering in pigs holds great promise in agriculture and biomedicine1. Using currently available approach, generating specific gene modifications in pigs requires two steps. First, site-specific nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are used to generate targeted mutations in pig somatic cells. Then t...
235 Citations Source Cite
Published on Jul 1, 2014in Cell Stem Cell 23.29
Adrian Veres10
Estimated H-index: 10
(Harvard University),
Bridget S. Gosis3
Estimated H-index: 3
(Harvard University)
+ 7 AuthorsKiran Musunuru49
Estimated H-index: 49
Summary Genome editing has attracted wide interest for the generation of cellular models of disease using human pluripotent stem cells and other cell types. CRISPR-Cas systems and TALENs can target desired genomic sites with high efficiency in human cells, but recent publications have led to concern about the extent to which these tools may cause off-target mutagenic effects that could potentially confound disease-modeling studies. Using CRISPR-Cas9 and TALEN targeted human pluripotent stem cell...
272 Citations Source Cite
Published on Jul 1, 2014in Cell Stem Cell 23.29
Keiichiro Suzuki15
Estimated H-index: 15
(Salk Institute for Biological Studies),
Chang Yu22
Estimated H-index: 22
+ 20 AuthorsEmi Aizawa10
Estimated H-index: 10
(Salk Institute for Biological Studies)
The utility of genome editing technologies for disease modeling and developing cellular therapies has been extensively documented, but the impact of these technologies on mutational load at the whole-genome level remains unclear. We performed whole-genome sequencing to evaluate the mutational load at single-base resolution in individual gene-corrected human induced pluripotent stem cell (hiPSC) clones in three different disease models. In single-cell clones, gene correction by helper-dependent a...
107 Citations Source Cite
Published on Nov 1, 2014in Nature 41.58
Havva Keskin5
Estimated H-index: 5
,
Ying Shen12
Estimated H-index: 12
+ 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.
108 Citations Source Cite
Published on Nov 1, 2013in Nature Protocols 12.42
F. Ann Ran10
Estimated H-index: 10
,
Patrick Hsu15
Estimated H-index: 15
+ 3 AuthorsFeng Zhang101
Estimated H-index: 101
Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed rep...
2,717 Citations Source Cite
Masahiro Onozawa16
Estimated H-index: 16
(National Institutes of Health),
Zhenhua Zhang7
Estimated H-index: 7
(National Institutes of Health)
+ 5 AuthorsPeter D. Aplan45
Estimated H-index: 45
(National Institutes of Health)
We show that DNA double-strand breaks (DSBs) can be repaired by insertion of 50- to 1,000-bp sequences termed “templated-sequence insertions” (TSIs) derived from distant regions of the genome. Additional experiments indicate that the source of template for repair was primarily nuclear RNA. This mode of DNA-DSB repair by insertion is not restricted to experimentally produced breaks but also occurs at the site of spontaneous DNA DSBs in human cells. These TSIs are polymorphic in the human genome, ...
31 Citations Source Cite
Published on Feb 1, 2014in Cell 31.40
Yuyu Niu15
Estimated H-index: 15
,
Bin Shen14
Estimated H-index: 14
(National Resource Center)
+ 25 AuthorsWei Li10
Estimated H-index: 10
(Chinese Academy of Sciences)
Summary Monkeys serve as important model species for studying human diseases and developing therapeutic strategies, yet the application of monkeys in biomedical researches has been significantly hindered by the difficulties in producing animals genetically modified at the desired target sites. Here, we first applied the CRISPR/Cas9 system, a versatile tool for editing the genes of different organisms, to target monkey genomes. By coinjection of Cas9 mRNA and sgRNAs into one-cell-stage embryos, w...
566 Citations Source Cite
Published on Aug 1, 2014in Nature Protocols 12.42
Hui Yang16
Estimated H-index: 16
,
Haoyi Wang23
Estimated H-index: 23
,
Rudolf Jaenisch171
Estimated H-index: 171
Crispr/Cas technology is a quick and efficient method of modifying the genomes of a range of organisms. Here the Jaenisch laboratory provides a protocol for CRISPR/Cas-mediated genome modification of mice.
196 Citations Source Cite
Published on Mar 1, 2015in Nature Biotechnology 35.72
Richard L. Frock12
Estimated H-index: 12
,
Jiazhi Hu11
Estimated H-index: 11
+ 3 AuthorsFrederick W. Alt154
Estimated H-index: 154
(Harvard University)
An unbiased genome-wide method reveals on- and off-target DNA cleavage by TALEN and Cas9 nucleases by detecting chromosome translocation events.
282 Citations Source Cite
  • References (48)
  • Citations (115)
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Cited By115
Published on Aug 1, 2015in Mammalian Genome 2.69
Susan M. Bello6
Estimated H-index: 6
,
Cynthia L. Smith20
Estimated H-index: 20
,
Janan T. Eppig45
Estimated H-index: 45
A core part of the Mouse Genome Informatics (MGI) resource is the collection of mouse mutations and the annotation phenotypes and diseases displayed by mice carrying these mutations. These data are integrated with the rest of data in MGI and exported to numerous other resources. The use of mouse phenotype data to drive translational research into human disease has expanded rapidly with the improvements in sequencing technology. MGI has implemented many improvements in allele and phenotype data a...
15 Citations Source Cite
Published on Dec 1, 2015in Genome Biology 13.21
William C. Skarnes47
Estimated H-index: 47
(Wellcome Trust)
Injection of recombinant Cas9 protein and synthetic guide RNAs into mouse zygotes has been shown to facilitate gene disruption and knock-ins using the CRISPR system. These technologies may soon displace genetic modification using embryonic stem cells.
11 Citations Source Cite
Published on Oct 1, 2015in Mammalian Genome 2.69
Janan T. Eppig45
Estimated H-index: 45
,
Howie Motenko5
Estimated H-index: 5
+ 2 AuthorsCynthia L. Smith20
Estimated H-index: 20
The availability of and access to quality genetically defined, health-status known mouse resources is critical for biomedical research. By ensuring that mice used in research experiments are biologically, genetically, and health-status equivalent, we enable knowledge transfer, hypothesis building based on multiple data streams, and experimental reproducibility based on common mouse resources (reagents). Major repositories for mouse resources have developed over time and each has significant uniq...
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Published on Dec 1, 2015in Genome Medicine 8.90
Haiwei Mou8
Estimated H-index: 8
(University of Massachusetts Medical School),
Zachary Kennedy3
Estimated H-index: 3
(University of Massachusetts Medical School)
+ 2 AuthorsWen Xue26
Estimated H-index: 26
(University of Massachusetts Medical School)
The cancer genome is highly complex, with hundreds of point mutations, translocations, and chromosome gains and losses per tumor. To understand the effects of these alterations, precise models are needed. Traditional approaches to the construction of mouse models are time-consuming and laborious, requiring manipulation of embryonic stem cells and multiple steps. The recent development of the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 system, a powerful genome-editin...
42 Citations Source Cite
Published on Dec 1, 2015in Epigenetics & Chromatin 5.35
Benjamin I. Laufer11
Estimated H-index: 11
(University of Western Ontario),
Shiva M. Singh31
Estimated H-index: 31
(University of Western Ontario)
Genome editing technology has evolved rather quickly and become accessible to most researchers. It has resulted in far reaching implications and a number of novel designer systems including epigenome editing. Epigenome editing utilizes a combination of nuclease-null genome editing systems and effector domains to modulate gene expression. In particular, Zinc Finger, Transcription-Activator-Like Effector, and CRISPR/Cas9 have emerged as modular systems that can be modified to allow for precision m...
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Published on Oct 1, 2015in Trends in Molecular Medicine 11.02
Lukas E. Dow22
Estimated H-index: 22
(Cornell University)
The recent advent of CRISPR/Cas9-mediated genome editing has created a wave of excitement across the scientific research community, carrying the promise of simple and effective genomic manipulation of nearly any cell type. CRISPR has quickly become the preferred tool for genetic manipulation, and shows incredible promise as a platform for studying gene function in vivo . I discuss the current application of CRISPR technology to create new in vivo disease models, with a particular focus on how th...
45 Citations Source Cite
Published on Sep 1, 2015in Genetics 4.08
Alexandre Paix12
Estimated H-index: 12
(Johns Hopkins University School of Medicine),
Andrew Folkmann3
Estimated H-index: 3
(Johns Hopkins University School of Medicine)
+ 1 AuthorsGeraldine Seydoux48
Estimated H-index: 48
(Johns Hopkins University School of Medicine)
Homology-directed repair (HDR) of breaks induced by the RNA-programmed nuclease Cas9 has become a popular method for genome editing in several organisms. Most HDR protocols rely on plasmid-based expression of Cas9 and the gene-specific guide RNAs. Here we report that direct injection of in vitro–assembled Cas9-CRISPR RNA (crRNA) trans-activating crRNA (tracrRNA) ribonucleoprotein complexes into the gonad of Caenorhabditis elegans yields HDR edits at a high frequency. Building on our earlier find...
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Published on Jul 1, 2015in Cell Research 15.39
Xiangyu Guo10
Estimated H-index: 10
,
Xiao-Jiang Li61
Estimated H-index: 61
Mosaic mutations and off-target effects caused by CRISPR/Cas9 have led to concerns about the efficiency and specificity of this new technique in non-human primates and other large animals. Here we discuss recent findings from primate embryos, with a focus on the technical issues CRISPR/Cas9 faces before producing non-human primate models of human diseases.
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Published on Feb 1, 2016in Cancer Letters 6.49
Ziguo Yang4
Estimated H-index: 4
(Shandong University),
Xiaobo Guo8
Estimated H-index: 8
(Shandong University)
+ 2 AuthorsLeping Li7
Estimated H-index: 7
(Shandong University)
Gastric cancer (GC) is a major threat to human health, and its prognosis is poor due to the lack of appropriate biomarkers. LncRNAs are a group of non-protein-coding RNAs that regulate gene expression at the transcriptional or posttranscriptional level. LncRNAs play essential roles in GC initiation and development in the same way as oncogenes or tumour suppressor genes. Recent investigations have revealed that lncRNAs are often aberrantly expressed in GC; are involved in cell proliferation, apop...
16 Citations Source Cite
Published on Dec 1, 2015in Transgenic Research 2.20
Satoshi Yamamoto8
Estimated H-index: 8
(Takeda Pharmaceutical Company),
Yuki Ooshima2
Estimated H-index: 2
(Takeda Pharmaceutical Company)
+ 7 AuthorsMichiyasu Takeyama4
Estimated H-index: 4
(Takeda Pharmaceutical Company)
The relative proportion of kynurenine aminotransferase (KAT) I–IV activities in the brain is similar between humans and rats. Moreover, KAT II is considered to be the main enzyme for kynurenic acid production in the brain. Taken together, human KAT II knock-in (hKAT II KI) rats will become a valuable tool for the evaluation of KAT II targeted drugs as a human mimetic model. Although we initially tried the approach by conventional gene-targeting via embryonic stem cells (ESCs) to generate them, w...
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