scholarly journals Application of CRISPR/Cas9 nuclease in amphioxus genome editing

2020 ◽  
Author(s):  
Liuru Su ◽  
Chenggang Shi ◽  
Xin Huang ◽  
Yiquan Wang ◽  
Guang Li
Keyword(s):  
Animal Model ◽  
Thermal Treatment ◽  
Genome Editing ◽  
Phylogenetic Position ◽  
Transcription Activator ◽  
Cell Stage ◽  
System A ◽  
Cas9 Nuclease ◽  
Cas9 Protein ◽  
Amphioxus Genome

AbstractThe cephalochordate amphioxus is a promising animal model for studying the origin of vertebrate due to its key phylogenetic position among chordates. Although transcription activator-like effector nucleases (TALENs) has been adopted in amphioxus genome editing, its labor intensive construction of the TALEN proteins limits its usage in many laboratories. We here report an application of the CRISPR/Cas9 system, a more amenable genome editing method, in this group of animals. Our data show that while co-injection of Cas9 mRNAs and sgRNAs into amphioxus unfertilized eggs causes no detectable mutations at targeted loci, injections of Cas9 mRNAs and sgRNAs at two cell stage, or of Cas9 protein and sgRNAs before fertilization can executes efficient disruptions of targeted genes. Among the nine tested sgRNAs (targeting five genes) co-injected with Cas9 protein, seven introduced mutations at efficiency ranging from 18.4% to 90% and four caused specific phenotypes in the injected embryos. We also demonstrate that monomerization of sgRNAs via thermal treatment or integration of sgRNAs into a longer backbone could increase mutation efficacies. Our study will not only promote application of genome editing method in amphioxus research, but also provide valuable experiences for other organisms in which the CRISPR/Cas9 system has not been successfully applied.

scholarly journals Application of CRISPR/Cas9 Nuclease in Amphioxus Genome Editing

Genes ◽  
2020 ◽  
Vol 11 (11) ◽  
pp. 1311
Author(s):  
Liuru Su ◽  
Chenggang Shi ◽  
Xin Huang ◽  
Yiquan Wang ◽  
Guang Li
Keyword(s):  
Animal Model ◽  
Thermal Treatment ◽  
Genome Editing ◽  
Phylogenetic Position ◽  
Transcription Activator ◽  
Cell Stage ◽  
System A ◽  
Cas9 Nuclease ◽  
Cas9 Protein ◽  
Amphioxus Genome

The cephalochordate amphioxus is a promising animal model for studying the origin of vertebrates due to its key phylogenetic position among chordates. Although transcription activator-like effector nucleases (TALENs) have been adopted in amphioxus genome editing, its labor-intensive construction of TALEN proteins limits its usage in many laboratories. Here we reported an application of the CRISPR/Cas9 system, a more amenable genome editing method, in this group of animals. Our data showed that while co-injection of Cas9 mRNAs and sgRNAs into amphioxus unfertilized eggs caused no detectable mutations at targeted loci, injections of Cas9 mRNAs and sgRNAs at the two-cell stage, or of Cas9 protein and sgRNAs before fertilization, can execute efficient disruptions of targeted genes. Among the nine tested sgRNAs (targeting five genes) co-injected with Cas9 protein, seven introduced mutations with efficiency ranging from 18.4% to 90% and four caused specific phenotypes in the injected embryos. We also demonstrated that monomerization of sgRNAs via thermal treatment or modifying the sgRNA structure could increase mutation efficacies. Our study will not only promote application of genome editing method in amphioxus research, but also provide valuable experiences for other organisms in which the CRISPR/Cas9 system has not been successfully applied.

scholarly journals The Relationship between Embryonic Development and the Efficiency of Target Mutations in Porcine Endogenous Retroviruses (PERVs) Pol Genes in Porcine Embryos

Animals ◽  
2019 ◽  
Vol 9 (9) ◽  
pp. 593 ◽  
Author(s):  
Maki Hirata ◽  
Manita Wittayarat ◽  
Takayuki Hirano ◽  
Nhien Thi Nguyen ◽  
Quynh Anh Le ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Genome Editing ◽  
Endogenous Retrovirus ◽  
Endogenous Retroviruses ◽  
Cell Stage ◽  
Gene Target ◽  
Porcine Endogenous Retrovirus ◽  
Cas9 Protein ◽  
Pig Genome ◽  
The Relationship

Porcine endogenous retrovirus (PERV) is a provirus found in the pig genome that may act as an infectious pathogen in humans who receive pig organ xenotransplantation. Inactivation of the PERV pol gene in porcine cells reportedly affects cell growth. Therefore, the mutation of PERV pol gene in porcine embryos using genome editing may affect the embryonic development. The present study was carried out to investigate the relationship between the mutation of the PERV pol gene in porcine embryos and their development. We introduced, either alone or in combination, three different gRNAs (gRNA1, 2, and 3) into porcine zygotes by genome editing using electroporation of the Cas9 protein (GEEP) system. All three gRNAs targeted the PERV pol gene, and we assessed their effects on porcine embryonic development. Our results showed that the blastocyst formation rates of zygotes electroporated with gRNA3—alone and in combination—were significantly lower (p < 0.05) than those of zygotes electroporated with gRNA1. The mutation rates assessed by the PERV pol gene target site sequencing in individual blastocysts and pooled embryos at the 2-to-8-cell stage did not differ among the three gRNAs. However, the frequency of indel mutations in mutant embryos at the 2-to-8-cell stage trended higher in the embryos electroporated with gRNA3 alone and in combination. Embryonic development may be affected by gRNAs that induce high-frequency indel mutations.

scholarly journals Various Aspects of a Gene Editing System—CRISPR–Cas9

2020 ◽  
Vol 21 (24) ◽  
pp. 9604
Author(s):  
Edyta Janik ◽  
Marcin Niemcewicz ◽  
Michal Ceremuga ◽  
Lukasz Krzowski ◽  
Joanna Saluk-Bijak ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Genome Engineering ◽  
High Efficiency ◽  
Adaptive Immune System ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Guide Rna ◽  
Adaptive Immune ◽  
Cas9 Protein

The discovery of clustered, regularly interspaced short palindromic repeats (CRISPR) and their cooperation with CRISPR-associated (Cas) genes is one of the greatest advances of the century and has marked their application as a powerful genome engineering tool. The CRISPR–Cas system was discovered as a part of the adaptive immune system in bacteria and archaea to defend from plasmids and phages. CRISPR has been found to be an advanced alternative to zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) for gene editing and regulation, as the CRISPR–Cas9 protein remains the same for various gene targets and just a short guide RNA sequence needs to be altered to redirect the site-specific cleavage. Due to its high efficiency and precision, the Cas9 protein derived from the type II CRISPR system has been found to have applications in many fields of science. Although CRISPR–Cas9 allows easy genome editing and has a number of benefits, we should not ignore the important ethical and biosafety issues. Moreover, any tool that has great potential and offers significant capabilities carries a level of risk of being used for non-legal purposes. In this review, we present a brief history and mechanism of the CRISPR–Cas9 system. We also describe on the applications of this technology in gene regulation and genome editing; the treatment of cancer and other diseases; and limitations and concerns of the use of CRISPR–Cas9.

scholarly journals Evolution and Application of Genome Editing Techniques for Achieving Food and Nutritional Security

2021 ◽  
Vol 22 (11) ◽  
pp. 5585
Author(s):  
Sajid Fiaz ◽  
Sunny Ahmar ◽  
Sajjad Saeed ◽  
Aamir Riaz ◽  
Freddy Mora-Poblete ◽  
...  
Keyword(s):  
Abiotic Stress ◽  
Food Security ◽  
Plant Breeding ◽  
Genome Editing ◽  
Crop Improvement ◽  
Hybrid Seed Production ◽  
Biotic And Abiotic Stress ◽  
Transcription Activator ◽  
Protein System ◽  
Breeding Techniques

A world with zero hunger is possible only through a sustainable increase in food production and distribution and the elimination of poverty. Scientific, logistical, and humanitarian approaches must be employed simultaneously to ensure food security, starting with farmers and breeders and extending to policy makers and governments. The current agricultural production system is facing the challenge of sustainably increasing grain quality and yield and enhancing resistance to biotic and abiotic stress under the intensifying pressure of climate change. Under present circumstances, conventional breeding techniques are not sufficient. Innovation in plant breeding is critical in managing agricultural challenges and achieving sustainable crop production. Novel plant breeding techniques, involving a series of developments from genome editing techniques to speed breeding and the integration of omics technology, offer relevant, versatile, cost-effective, and less time-consuming ways of achieving precision in plant breeding. Opportunities to edit agriculturally significant genes now exist as a result of new genome editing techniques. These range from random (physical and chemical mutagens) to non-random meganucleases (MegaN), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein system 9 (CRISPR/Cas9), the CRISPR system from Prevotella and Francisella1 (Cpf1), base editing (BE), and prime editing (PE). Genome editing techniques that promote crop improvement through hybrid seed production, induced apomixis, and resistance to biotic and abiotic stress are prioritized when selecting for genetic gain in a restricted timeframe. The novel CRISPR-associated protein system 9 variants, namely BE and PE, can generate transgene-free plants with more frequency and are therefore being used for knocking out of genes of interest. We provide a comprehensive review of the evolution of genome editing technologies, especially the application of the third-generation genome editing technologies to achieve various plant breeding objectives within the regulatory regimes adopted by various countries. Future development and the optimization of forward and reverse genetics to achieve food security are evaluated.

scholarly journals An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

2021 ◽  
Author(s):  
Eugene V. Gasanov ◽  
Justyna Jędrychowska ◽  
Michal Pastor ◽  
Malgorzata Wiweger ◽  
Axel Methner ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Target Site ◽  
Proof Of Concept ◽  
Intervention Site ◽  
End Joining ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Non Homologous End Joining

AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

scholarly journals Evaluating the Efficiency of gRNAs in CRISPR/Cas9 Mediated Genome Editing in Poplars

2019 ◽  
Vol 20 (15) ◽  
pp. 3623 ◽  
Author(s):  
Tobias Bruegmann ◽  
Khira Deecke ◽  
Matthias Fladung
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Constitutive Expression ◽  
Gc Content ◽  
Seed Region ◽  
Research Topics ◽  
Target Region ◽  
Cas9 Nuclease ◽  
Different Types ◽  
Poplar Hybrid

CRISPR/Cas9 has become one of the most promising techniques for genome editing in plants and works very well in poplars with an Agrobacterium-mediated transformation system. We selected twelve genes, including SOC1, FUL, and their paralogous genes, four NFP-like genes and TOZ19 for three different research topics. The gRNAs were designed for editing, and, together with a constitutively expressed Cas9 nuclease, transferred either into the poplar hybrid Populus × canescens or into P. tremula. The regenerated lines showed different types of editing and revealed several homozygous editing events which are of special interest in perennial species because of limited back-cross ability. Through a time series, we could show that despite the constitutive expression of the Cas9 nuclease, no secondary editing of the target region occurred. Thus, constitutive Cas9 expression does not seem to pose any risk to additional editing events. Based on various criteria, we obtained evidence for a relationship between the structure of gRNA and the efficiency of gene editing. In particular, the GC content, purine residues in the gRNA end, and the free accessibility of the seed region seemed to be highly important for genome editing in poplars. Based on our findings on nine different poplar genes, efficient gRNAs can be designed for future efficient editing applications in poplars.

scholarly journals Production of α1,3-galactosyltransferase targeted pigs using transcription activator-like effector nuclease-mediated genome editing technology

2016 ◽  
Vol 17 (1) ◽  
pp. 89 ◽  
Author(s):  
Jung-Taek Kang ◽  
Dae-Kee Kwon ◽  
A-Rum Park ◽  
Eun-Jin Lee ◽  
Yun-Jin Yun ◽  
...  
Keyword(s):  
Genome Editing ◽  
Transcription Activator

scholarly journals TALEN and CRISPR/Cas Genome Editing Systems: Tools of Discovery

Acta Naturae ◽  
2014 ◽  
Vol 6 (3) ◽  
pp. 19-40 ◽  
Author(s):  
A. A. Nemudryi ◽  
K. R. Valetdinova ◽  
S. P. Medvedev ◽  
S. M. Zakian
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Genome Engineering ◽  
Disease Modeling ◽  
Regulation Of Gene Expression ◽  
Hereditary Disease ◽  
Transcription Activator ◽  
Wide Range ◽  
High Standards ◽  
Phenotype Formation

Precise studies of plant, animal and human genomes enable remarkable opportunities of obtained data application in biotechnology and medicine. However, knowing nucleotide sequences isnt enough for understanding of particular genomic elements functional relationship and their role in phenotype formation and disease pathogenesis. In post-genomic era methods allowing genomic DNA sequences manipulation, visualization and regulation of gene expression are rapidly evolving. Though, there are few methods, that meet high standards of efficiency, safety and accessibility for a wide range of researchers. In 2011 and 2013 novel methods of genome editing appeared - this are TALEN (Transcription Activator-Like Effector Nucleases) and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 systems. Although TALEN and CRISPR/Cas9 appeared recently, these systems have proved to be effective and reliable tools for genome engineering. Here we generally review application of these systems for genome editing in conventional model objects of current biology, functional genome screening, cell-based human hereditary disease modeling, epigenome studies and visualization of cellular processes. Additionally, we review general strategies for designing TALEN and CRISPR/Cas9 and analyzing their activity. We also discuss some obstacles researcher can face using these genome editing tools.

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