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CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein

Published on Jun 1, 2017in Molecular Genetics and Genomics 2.73
· DOI :10.1007/s00438-017-1299-z
Lichun Tang1
Estimated H-index: 1
(Protein Sciences),
Yanting Zeng1
Estimated H-index: 1
(Guangzhou Medical University)
+ 8 AuthorsJianqiao Liu5
Estimated H-index: 5
(Guangzhou Medical University)
Abstract
Previous works using human tripronuclear zygotes suggested that the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system could be a tool in correcting disease-causing mutations. However, whether this system was applicable in normal human (dual pronuclear, 2PN) zygotes was unclear. Here we demonstrate that CRISPR/Cas9 is also effective as a gene-editing tool in human 2PN zygotes. By injection of Cas9 protein complexed with the appropriate sgRNAs and homology donors into one-cell human embryos, we demonstrated efficient homologous recombination-mediated correction of point mutations in HBB and G6PD. However, our results also reveal limitations of this correction procedure and highlight the need for further research.
  • References (26)
  • Citations (80)
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References26
Newest
Published on Oct 1, 2016in Developmental Biology 3.26
Masakazu Hashimoto7
Estimated H-index: 7
(Osaka University),
Yukiko Yamashita2
Estimated H-index: 2
(University of Tokushima),
Tatsuya Takemoto13
Estimated H-index: 13
(University of Tokushima)
Abstract The CRISPR/Cas9 system is a powerful tool for elucidating the roles of genes in a wide variety of organisms including mice. To obtain genetically modified embryos or mice by this method, Cas9 mRNA and sgRNA are usually introduced into zygotes by microinjection or electroporation. However, most mutants generated with this method are genetically mosaic, composed of several types of cells carrying different mutations, which complicates phenotype analysis in founder embryos or mice. To simp...
47 Citations Source Cite
Published on Jul 8, 2016in Journal of Biological Chemistry 4.01
Sean Chen2
Estimated H-index: 2
(University of California, Berkeley),
Benjamin Lee1
Estimated H-index: 1
(University of California, Berkeley)
+ 2 AuthorsLin He27
Estimated H-index: 27
(University of California, Berkeley)
Abstract The CRISPR/Cas9 system has been employed to efficiently edit the genomes of diverse model organisms. CRISPR-mediated mouse genome editing is typically accomplished by microinjection of Cas9 DNA/RNA and single-guide RNA into zygotes to generate modified animals in one step. However, microinjection is a technically demanding, labor-intensive, and costly procedure with poor embryo viability. Here, we describe a simple and economic electroporation-based strategy to deliver Cas9/sgRNA ribonu...
65 Citations Source Cite
Published on May 1, 2016in Journal of Assisted Reproduction and Genetics 2.79
Xiangjin Kang6
Estimated H-index: 6
(Guangzhou Medical University),
Wenyin He7
Estimated H-index: 7
(Guangzhou Medical University)
+ 5 AuthorsYong Fan12
Estimated H-index: 12
(Guangzhou Medical University)
Purpose As a powerful technology for genome engineering, the CRISPR/Cas system has been successfully applied to modify the genomes of various species. The purpose of this study was to evaluate the technology and establish principles for the introduction of precise genetic modifications in early human embryos.
118 Citations Source Cite
Published on May 1, 2015in Protein & Cell 6.23
Puping Liang7
Estimated H-index: 7
(SYSU: Sun Yat-sen University),
Yanwen Xu14
Estimated H-index: 14
(SYSU: Sun Yat-sen University)
+ 13 AuthorsYujing Li10
Estimated H-index: 10
(SYSU: Sun Yat-sen University)
Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISP...
458 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 Jul 1, 2014in G3: Genes, Genomes, Genetics 2.74
Jeong-Soo Lee8
Estimated H-index: 8
(Korea Research Institute of Bioscience and Biotechnology),
Su-Jin Kwak3
Estimated H-index: 3
(Korea Research Institute of Bioscience and Biotechnology)
+ 4 AuthorsKweon Yu24
Estimated H-index: 24
(Korea Research Institute of Bioscience and Biotechnology)
We report a method for generating Drosophila germline mutants effectively via injection of the complex of the purified Cas9 protein, tracrRNA, and gene-specific crRNAs, which may reduce delayed mutations because of the transient activity of the Cas9 protein, combined with the simple mutation detection in GO founders by the T7E1 assay.
26 Citations Source Cite
Published on Jun 17, 2014in Nucleic Acids Research 11.56
Yanni Lin4
Estimated H-index: 4
(Georgia Institute of Technology),
Thomas J. Cradick18
Estimated H-index: 18
(Georgia Institute of Technology)
+ 7 AuthorsGang Bao56
Estimated H-index: 56
(Georgia Institute of Technology)
CRISPR/Cas9 systems are a versatile tool for genome editing due to the highly efficient targeting of DNA sequences complementary to their RNA guide strands. However, it has been shown that RNA-guided Cas9 nuclease cleaves genomic DNA sequences containing mismatches to the guide strand. A better understanding of the CRISPR/Cas9 specificity is needed to minimize off-target cleavage in large mammalian genomes. Here we show that genomic sites could be cleaved by CRISPR/Cas9 systems when DNA sequence...
259 Citations Source Cite
Published on Jun 1, 2014in Genome Research 10.10
Sojung Kim10
Estimated H-index: 10
(SNU: Seoul National University),
Daesik Kim12
Estimated H-index: 12
(SNU: Seoul National University)
+ 2 AuthorsJin-Soo Kim52
Estimated H-index: 52
(SNU: Seoul National University)
RNA-guided engineered nucleases (RGENs) derived from the prokaryotic adaptive immune system known as CRISPR (clustered, regularly interspaced, short palindromic repeat)/Cas (CRISPR-associated) enable genome editing in human cell lines, animals, and plants, but are limited by off-target effects and unwanted integration of DNA segments derived from plasmids encoding Cas9 and guide RNA at both on-target and off-target sites in the genome. Here, we deliver purified recombinant Cas9 protein and guide...
578 Citations Source Cite
Published on Mar 5, 2014in PLOS ONE 2.77
Yuanwu Ma6
Estimated H-index: 6
(Academy of Medical Sciences, United Kingdom),
Bin Shen3
Estimated H-index: 3
(National Resource Center)
+ 5 AuthorsLianfeng Zhang6
Estimated H-index: 6
(Academy of Medical Sciences, United Kingdom)
The CRISPR/Cas9 system has been proven to be an efficient gene-editing tool for genome modification of cells and organisms. Multiplex genetic engineering in rat holds a bright future for the study of complex disease. Here, we show that this system enables the simultaneous disruption of four genes (ApoE, B2m, Prf1, and Prkdc) in rats in one-step, by co-injection of Cas9 mRNA and sgRNAs into fertilized eggs. We further observed the gene modifications are germline transmittable, and confirmed the o...
61 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 Li41
Estimated H-index: 41
(CAS: 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...
584 Citations Source Cite
Cited By80
Newest
Published on May 23, 2019
Yagiz Aksoy1
Estimated H-index: 1
(Macquarie University),
David T. Nguyen1
Estimated H-index: 1
(Garvan Institute of Medical Research)
+ 4 AuthorsDaniel Hesselson14
Estimated H-index: 14
(UNSW: University of New South Wales)
Precise genome editing is limited by the inefficiency of homology-directed repair (HDR) compared to the non-homologous end-joining (NHEJ) of double strand breaks (DSBs). The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 system generates precise, locus-specific DSBs that can serve as substrates for HDR. We developed an in vivo visual reporter assay to quantify HDR-mediated events at single-cell resolution in zebrafish and used this system to identify small-molecule modula...
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Published on Nov 14, 2018in Protein & Cell 6.23
Yanni Li (Guangzhou Medical University), Xiang Jin Kang1
Estimated H-index: 1
(Guangzhou Medical University)
+ 3 AuthorsYong Fan12
Estimated H-index: 12
(Guangzhou Medical University)
Source Cite
Published on Jun 3, 2019in Nature Medicine 32.62
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Published on May 5, 2019in Qualitative Health Research 2.41
Kirsten A. Riggan (Mayo Clinic), Richard R. Sharp36
Estimated H-index: 36
(Mayo Clinic),
Megan Allyse6
Estimated H-index: 6
(Mayo Clinic)
The application of gene editing technologies to prevent or mitigate genetic disease in humans is considered one of its most promising applications. However, as the technology advances, it is imperative to understand the views of the broader public on how it should be used. We conducted focus groups to understand public views on the ethical permissibility and governance of gene editing technologies in humans. A total of 50 urban and semirural residents in the upper Midwest took part in six focus ...
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Published on May 1, 2019in Pflügers Archiv: European Journal of Physiology 2.77
Maksymilian Prondzynski6
Estimated H-index: 6
(UHH: University of Hamburg),
Giulia Mearini16
Estimated H-index: 16
(UHH: University of Hamburg),
Lucie Carrier43
Estimated H-index: 43
(UHH: University of Hamburg)
Hypertrophic cardiomyopathy (HCM) is an inherited myocardial disease with an estimated prevalence of 1:200 caused by mutations in sarcomeric proteins. It is associated with hypertrophy of the left ventricle, increased interstitial fibrosis, and diastolic dysfunction for heterozygous mutation carriers. Carriers of double heterozygous, compound heterozygous, and homozygous mutations often display more severe forms of cardiomyopathies, ultimately leading to premature death. So far, there is no cura...
1 Citations Source Cite
Published on Apr 3, 2019
Jennifer M. Gumer (LMU: Loyola Marymount University)
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