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Gene editing technologies, such as CRISPR-Cas9, have important applications in mammalian embryos for generating novel animal models in biomedical research and lines of livestock with enhanced production traits. However, the lack of methods for efficient introduction of gene editing reagents into zygotes of various species and the need for surgical embryo transfer in mice have been technical barriers of widespread use. Here, we described methodologies that overcome these limitations for embryos of mice, cattle, and pigs. Using mutation of the Nanos2 gene as a readout, we refined electroporation parameters with preassembled sgRNA-Cas9 RNPs for zygotes of all three species without the need for zona pellucida dissolution that led to high-efficiency INDEL edits. In addition, we optimized culture conditions to support maturation from zygote to the multicellular stage for all three species that generates embryos ready for transfer to produce gene-edited animals. Moreover, for mice, we devised a nonsurgical embryo transfer method that yields offspring at an efficiency comparable to conventional surgical approaches. Collectively, outcomes of these studies provide simplified pipelines for CRISPR-Cas9-based gene editing that are applicable in a variety of mammalian species.
This article was published in the following journal.
Name: Biology of reproduction
The genome editors CRISPR/Cas9 (clustered regularly interspaced short palindromicrepeats/Cas9 nuclease-null) and TALENs (transcription activator-like effector nuclease) are popularly used for targeted...
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated nuclease 9 (Cas9), as a powerful genome-editing tool, has revolutionized genetic engineering. It is widely used to ...
The CRISPR/Cas9 system is a rapid, simple, and often extremely efficient gene editing method. This method has been used in a variety of organisms and cell types over the past several years. However, u...
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This is an open-label and triple cohort study of the safety and efficacy of TALEN and CRISPR/Cas9 to possibly treat HPV Persistency and human cervical intraepithelial neoplasiaⅠwithout i...
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Multiple solid tumors have positive targets of mesothelin expressed on the surfaces of the tumor cells, we use the technique of CRISPR-Cas9 to knocked out the PD-1 and TCR of chimeric anti...
The purpose of the current study is to evaluate the effect of preimplantation genetic screening (PGS) by next generation sequencing (NGS) compared to standard morphological assessment of e...
This is a single centre、single arm、open-label study，to investigate the safety and efficacy of the gene correction of HBB in patient-specific iHSCs using CRISPR/Cas9.
Protein components of the CRISPR-CAS SYSTEMS for anti-viral defense in ARCHAEA and BACTERIA. These are proteins that carry out a variety of functions during the creation and expansion of the CRISPR ARRAYS, the capture of new CRISPR SPACERS, biogenesis of SMALL INTERFERING RNA (CRISPR or crRNAs), and the targeting and silencing of invading viruses and plasmids. They include DNA HELICASES; RNA-BINDING PROTEINS; ENDONUCLEASES; and RNA and DNA POLYMERASES.
Adaptive antiviral defense mechanisms, in archaea and bacteria, based on DNA repeat arrays called CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR elements) that function in conjunction with CRISPR-ASSOCIATED PROTEINS (Cas proteins). Several types have been distinguished, including Type I, Type II, and Type III, based on signature motifs of CRISPR-ASSOCIATED PROTEINS.
Major egg yolk proteins from egg-laying animals such as non-mammalian VERTEBRATES; ARTHROPODS; and others. They are high-density lipoglycoproteins derived from circulating precursors, VITELLOGENINS. Vitellins serve as nutrients for the growing non-mammalian embryos (EMBRYO, NONMAMMALIAN).
The transfer of mammalian embryos from an in vivo or in vitro environment to a suitable host to improve pregnancy or gestational outcome in human or animal. In human fertility treatment programs, preimplantation embryos ranging from the 4-cell stage to the blastocyst stage are transferred to the uterine cavity between 3-5 days after FERTILIZATION IN VITRO.
A genus of STARFISH in the family Asterinidae. They externally hold developing embryos (EMBRYO, NON-MAMMALIAN) among the spines below the oral surface.
Genetic engineering is the process of manually adding new DNA to an organism. The goal is to add one or more new traits that are not already found in that organism. Examples of genetically engineered (transgenic) organisms currently on the market include...
Bioinformatics is the application of computer software and hardware to the management of biological data to create useful information. Computers are used to gather, store, analyze and integrate biological and genetic information which can then be applied...