scholarly journals Precision genome editing in plants via gene targeting and piggy B ac ‐mediated marker excision

2014 ◽  
Vol 81 (1) ◽  
pp. 160-168 ◽  
Author(s):  
Ayako Nishizawa‐Yokoi ◽  
Masaki Endo ◽  
Namie Ohtsuki ◽  
Hiroaki Saika ◽  
Seiichi Toki
Keyword(s):  
Genome Editing ◽  
Gene Targeting ◽  
Marker Excision

scholarly journals Precise Genome Editing in miRNA Target Site via Gene Targeting and Subsequent Single-Strand-Annealing-Mediated Excision of the Marker Gene in Plants

2021 ◽  
Vol 2 ◽  
Author(s):  
Namie Ohtsuki ◽  
Keiko Kizawa ◽  
Akiko Mori ◽  
Ayako Nishizawa-Yokoi ◽  
Takao Komatsuda ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Targeting ◽  
Mirna Target ◽  
Single Strand ◽  
Target Site ◽  
Selection Marker ◽  
Base Substitution ◽  
Marker Excision ◽  
Single Strand Annealing ◽  
Strand Annealing

Gene targeting (GT) enables precise genome modification—e.g., the introduction of base substitutions—using donor DNA as a template. Combined with clean excision of the selection marker used to select GT cells, GT is expected to become a standard, generally applicable, base editing system. Previously, we demonstrated marker excision via a piggyBac transposon from GT-modified loci in rice. However, piggyBac-mediated marker excision has the limitation that it recognizes only the sequence TTAA. Recently, we proposed a novel and universal precise genome editing system consisting of GT with subsequent single-strand annealing (SSA)-mediated marker excision, which has, in principle, no limitation of target sequences. In this study, we introduced base substitutions into the microRNA miR172 target site of the OsCly1 gene—an ortholog of the barley Cleistogamy1 gene involved in cleistogamous flowering. To ensure efficient SSA, the GT vector harbors 1.2-kb overlapped sequences at both ends of a selection marker. The frequency of positive–negative selection-mediated GT using the vector with overlapped sequences was comparable with that achieved using vectors for piggyBac-mediated marker excision without overlapped sequences, with the frequency of SSA-mediated marker excision calculated as ~40% in the T0 generation. This frequency is thought to be adequate to produce marker-free cells, although it is lower than that achieved with piggyBac-mediated marker excision, which approaches 100%. To date, introduction of precise substitutions in discontinuous multiple bases of a targeted gene using base editors and the prime editing system based on CRISPR/Cas9 has been quite difficult. Here, using GT and our SSA-mediated marker excision system, we succeeded in the precise base substitution not only of single bases but also of artificial discontinuous multiple bases in the miR172 target site of the OsCly1 gene. Precise base substitution of miRNA target sites in target genes using this precise genome editing system will be a powerful tool in the production of valuable crops with improved traits.

scholarly journals Improved gene targeting in C. elegans using counter-selection and Flp-mediated marker excision

Genomics ◽  
2010 ◽  
Vol 95 (1) ◽  
pp. 37-46 ◽  
Author(s):  
Rafael P. Vázquez-Manrique ◽  
James C. Legg ◽  
Birgitta Olofsson ◽  
Sung Ly ◽  
Howard A. Baylis
Keyword(s):  
Gene Targeting ◽  
C Elegans ◽  
Counter Selection ◽  
Marker Excision

scholarly journals CRISPR/Cas9 Genome Editing Technology: A Valuable Tool for Understanding Plant Cell Wall Biosynthesis and Function

2020 ◽  
Vol 11 ◽  
Author(s):  
Yuan Zhang ◽  
Allan M. Showalter
Keyword(s):  
Cell Wall ◽  
Genome Editing ◽  
Gene Targeting ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Cell Structure ◽  
Higher Order ◽  
Knock Out ◽  
Genetic Crossing ◽  
And Function

For the past 5 years, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology has appeared in the molecular biology research spotlight. As a game-changing player in genome editing, CRISPR/Cas9 technology has revolutionized animal research, including medical research and human gene therapy as well as plant science research, particularly for crop improvement. One of the most common applications of CRISPR/Cas9 is to generate genetic knock-out mutants. Recently, several multiplex genome editing approaches utilizing CRISPR/Cas9 were developed and applied in various aspects of plant research. Here we summarize these approaches as they relate to plants, particularly with respect to understanding the biosynthesis and function of the plant cell wall. The plant cell wall is a polysaccharide-rich cell structure that is vital to plant cell formation, growth, and development. Humans are heavily dependent on the byproducts of the plant cell wall such as shelter, food, clothes, and fuel. Genes involved in the assembly of the plant cell wall are often highly redundant. To identify these redundant genes, higher-order knock-out mutants need to be generated, which is conventionally done by genetic crossing. Compared with genetic crossing, CRISPR/Cas9 multi-gene targeting can greatly shorten the process of higher-order mutant generation and screening, which is especially useful to characterize cell wall related genes in plant species that require longer growth time. Moreover, CRISPR/Cas9 makes it possible to knock out genes when null T-DNA mutants are not available or are genetically linked. Because of these advantages, CRISPR/Cas9 is becoming an ideal and indispensable tool to perform functional studies in plant cell wall research. In this review, we provide perspectives on how to design CRISPR/Cas9 to achieve efficient gene editing and multi-gene targeting in plants. We also discuss the recent development of the virus-based CRISPR/Cas9 system and the application of CRISPR/Cas9 to knock in genes. Lastly, we summarized current progress on using CRISPR/Cas9 for the characterization of plant cell wall-related genes.

scholarly journals Welcoming a new age for gene therapy in hematology

Blood ◽  
2016 ◽  
Vol 127 (21) ◽  
pp. 2523-2524 ◽  
Author(s):  
Mitchell J. Weiss ◽  
Charles G. Mullighan
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Genome Editing ◽  
Gene Targeting ◽  
Embryonic Stem ◽  
Hematological Disorders ◽  
Cellular Genes ◽  
Mammalian Genomes ◽  
Template Dna

Abstract Our capacities to understand and manipulate mammalian genomes are accelerating at an astounding pace. In 2007, Capecchi, Evans, and Smithies were awarded the Nobel Prize in medicine for their work on gene targeting, which showed that embryonic stem cells could be modified by homologous recombination (HR) with engineered template DNA to alter virtually any gene and create mutant mice. This work revolutionized biology by allowing investigators to study the in vivo consequences of selected gene alteration. However, the efficiency of HR in embryonic stem cells is unpredictable, depending on the target gene and HR template. More importantly, spontaneous HR occurs at very low rates in most somatic cells, restricting the use of standard gene targeting for most laboratory and clinical applications. This limitation is being overcome by genome-editing technologies, which markedly enhance the capacity to alter cellular genes with laser-like precision. Four review articles in this edition of Blood summarize the field of genome editing, focusing on its potential for treating hematological disorders.

scholarly journals In-planta Gene Targeting in Barley using Cas9, with and without Geminiviral Replicons

2021 ◽  
Author(s):  
Tom Lawrenson ◽  
Alison Hinchliffe ◽  
Martha Clarke ◽  
Yvie Morgan ◽  
Wendy Harwood
Keyword(s):  
Genome Editing ◽  
Gene Targeting ◽  
Target Gene ◽  
In Planta ◽  
Crop Species ◽  
Increase Copy Number ◽  
Independent Gene ◽  
Copy Numbers ◽  
Wide Range ◽  
Repair Template

AbstractAdvances in the use of RNA-guided Cas9-based genome editing in plants have been rapid over the last few years. A desirable application of genome editing is gene targeting (GT), as it allows a wide range of precise modifications, however this remains inefficient especially in key crop species. Here we describe successful, heritable gene targeting in barley using an in-planta strategy but fail to achieve the same using a wheat dwarf virus replicon to increase copy number of the repair template. Without the replicon, we were able to delete 150bp of the coding sequence of our target gene whilst simultaneously fusing in-frame mCherry in its place. Starting from 14 original transgenic plants, two plants appeared to have the required gene targeting event. From one of these T0 plants, three independent gene targeting events were identified, two of which were heritable. When the replicon was included, 39 T0 plants were produced and shown to have high copy numbers of the repair template. However, none of the 17 lines screened in T1 gave rise to significant or heritable gene targeting events despite screening twice the number of plants in T1 compared to the non-replicon strategy. Investigation indicated that high copy numbers of repair template created by the replicon approach cause false positive PCR results which are indistinguishable at the sequence level to true GT events in junction PCR screens widely used in GT studies. In the successful non-replicon approach, heritable gene targeting events were obtained in T1 and subsequently the T-DNA was found to be linked to the targeted locus. Thus, physical proximity of target and donor sites may be a factor in successful gene targeting.

Sign in / Sign up

Export Citation Format

Share Document