scholarly journals Efficient Targeted Gene Insertion in Maize using Agrobacterium-Mediated Delivery

2020 ◽  
Author(s):  
Sergei Svitashev ◽  
Dave Peterson ◽  
Pierjuigi Barone ◽  
Brian Lenderts ◽  
Chris Schwartz ◽  
...  
Keyword(s):  
Genome Editing ◽  
Strand Break ◽  
Plant Genome ◽  
Specific Gene ◽  
Crop Species ◽  
Gene Insertion ◽  
Maize Transformation ◽  
Dna Double Strand Break ◽  
Genomic Location ◽  
Marker Free

Abstract CRISPR-Cas is a powerful DNA double strand break technology with wide-ranging applications in plant genome modification. However, the efficiency of genome editing depends on various factors including plant genetic transformation processes and types of modifications desired. Agrobacterium infection is the preferred method of transformation and delivery of editing components into the plant cell. While this method has been successfully used to generate gene knock-outs in multiple crops, precise nucleotide replacement and especially gene insertion into a pre-defined genomic location remain highly challenging. Here we report an efficient, heritable, selectable marker-free site-specific gene insertion in maize using Agrobacterium-mediated delivery. Advancements in maize transformation and new vector design enabled targeted insertion with frequencies as high as 8–10%. Importantly, these advancements allowed not only an improvement of the frequency but also of the quality of generated events. These results further enable the application of genome editing for trait product development in a wide variety of crop species amenable to Agrobacterium-mediated transformation.

scholarly journals Base editing in Streptomyces with Cas9-deaminase fusions

2019 ◽  
Author(s):  
Zhiyu Zhong ◽  
Junhong Guo ◽  
Liang Deng ◽  
Li Chen ◽  
Jian Wang ◽  
...  
Keyword(s):  
Genome Editing ◽  
Target Genes ◽  
Genetic Manipulation ◽  
Strand Break ◽  
High Fidelity ◽  
Nucleotide Substitutions ◽  
Base Editing ◽  
Dna Double Strand Break ◽  
Repair Template ◽  
Adenine Base

AbstractConventional CRISPR/Cas genetic manipulation has been profitably applied to the genus Streptomyces, the most prolific bacterial producers of antibiotics. However, its reliance on DNA double-strand break (DSB) formation leads to unacceptably low yields of desired recombinants. We have adapted for Streptomyces recently-introduced cytidine base editors (CBEs) and adenine base editors (ABEs) which enable targeted C-to-T or A-to-G nucleotide substitutions, respectively, bypassing DSB and the need for a repair template. We report successful genome editing in Streptomyces at frequencies of around 50% using defective Cas9-guided base editors and up to 100% by using nicked Cas9-guided base editors. Furthermore, we demonstrate the multiplex genome editing potential of the nicked Cas9-guided base editor BE3 by programmed mutation of nine target genes simultaneously. Use of the high-fidelity version of BE3 (HF-BE3) essentially improved editing specificity. Collectively, this work provides a powerful new tool for genome editing in Streptomyces.

Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance

2020 ◽  
Author(s):  
Ruben Schep ◽  
Eva K. Brinkman ◽  
Christ Leemans ◽  
Xabier Vergara ◽  
Ben Morris ◽  
...  
Keyword(s):  
Genome Editing ◽  
Strand Break ◽  
Double Strand Break ◽  
Repair Pathway ◽  
End Joining ◽  
Dsb Repair ◽  
Dna Double Strand Break ◽  
Repair Pathways ◽  
Non Homologous End Joining ◽  
The Impact

AbstractDNA double-strand break (DSB) repair is mediated by multiple pathways, including classical non-homologous end-joining pathway (NHEJ) and several homology-driven repair pathways. This is particularly important for Cas9-mediated genome editing, where the outcome critically depends on the pathway that repairs the break. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a newly developed multiplexed reporter assay in combination with Cas9 cutting, we systematically measured the relative activities of three DSB repair pathways as function of chromatin context in >1,000 genomic locations. This revealed that NHEJ is broadly biased towards euchromatin, while microhomology-mediated end-joining (MMEJ) is more efficient in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 shifts the balance towards NHEJ. Single-strand templated repair (SSTR), often used for precise CRISPR editing, competes with MMEJ, and this competition is weakly associated with chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance, and guidance for the design of Cas9-mediated genome editing experiments.

scholarly journals Deletion-bias in DNA double-strand break repair differentially contributes to plant genome shrinkage

2017 ◽  
Vol 214 (4) ◽  
pp. 1712-1721 ◽  
Author(s):  
Giang T. H. Vu ◽  
Hieu X. Cao ◽  
Bernd Reiss ◽  
Ingo Schubert
Keyword(s):  
Strand Break ◽  
Double Strand Break ◽  
Plant Genome ◽  
Double Strand Break Repair ◽  
Dna Double Strand Break ◽  
Break Repair

scholarly journals Rheostatic Control of Cas9-Mediated DNA Double Strand Break (DSB) Generation and Genome Editing

2017 ◽  
Vol 13 (2) ◽  
pp. 438-442 ◽  
Author(s):  
John C. Rose ◽  
Jason J. Stephany ◽  
Cindy T. Wei ◽  
Douglas M. Fowler ◽  
Dustin J. Maly
Keyword(s):  
Genome Editing ◽  
Strand Break ◽  
Double Strand Break ◽  
Dna Double Strand Break

scholarly journals CRISPR/Cas9 Mediated Genome Editing in Crop Plants

2022 ◽  
Vol 9 (sp) ◽  
pp. 2396-2400
Author(s):  
Abdulrezzak Memon
Keyword(s):  
Genome Editing ◽  
New Technologies ◽  
Genetic Research ◽  
Herbicide Tolerance ◽  
Plant Genome ◽  
Genomic Research ◽  
Biotic And Abiotic Stress ◽  
Crop Species ◽  
Research Areas ◽  
Target Dna

Recently, most genomic research has focused on genome editing methods to develop new technologies that could be easy, reliable, and feasible to edit plant genomes for highly productive agriculture. Genome editing is based on alternating a specific target DNA sequence by adding, replacing, and removing DNA bases. This newest technology called CRISPR/Cas9 seems to be less time-consuming, more effective and used in many research areas of plant genetic research. CRISPR/Cas9 systems have many advantages in comparison with ZFNs and TALENs and has been extensively used for genome editing to many crop plant species. Around 20 crop species are successfully worked out for trait improvements, for example, yield improvement, disease resistance, herbicide tolerance, and biotic and abiotic stress management. This review paper will overview recent advances in CRISPR/Cas genome editing research in detail. The main focus will be on the use of CRISPR/Cas9 technology in plant genome research.

scholarly journals Non-GM Genome Editing Approaches in Crops

2021 ◽  
Vol 3 ◽  
Author(s):  
Zheng Gong ◽  
Ming Cheng ◽  
Jose R. Botella
Keyword(s):  
Genome Editing ◽  
Viral Vectors ◽  
Large Scale ◽  
First Generation ◽  
Transformation Process ◽  
Plant Genome ◽  
Crop Species ◽  
Breeding Programs ◽  
Public Controversy ◽  
Rigid Cell Wall

CRISPR/Cas-based genome editing technologies have the potential to fast-track large-scale crop breeding programs. However, the rigid cell wall limits the delivery of CRISPR/Cas components into plant cells, decreasing genome editing efficiency. Established methods, such as Agrobacterium tumefaciens-mediated or biolistic transformation have been used to integrate genetic cassettes containing CRISPR components into the plant genome. Although efficient, these methods pose several problems, including 1) The transformation process requires laborious and time-consuming tissue culture and regeneration steps; 2) many crop species and elite varieties are recalcitrant to transformation; 3) The segregation of transgenes in vegetatively propagated or highly heterozygous crops, such as pineapple, is either difficult or impossible; and 4) The production of a genetically modified first generation can lead to public controversy and onerous government regulations. The development of transgene-free genome editing technologies can address many problems associated with transgenic-based approaches. Transgene-free genome editing have been achieved through the delivery of preassembled CRISPR/Cas ribonucleoproteins, although its application is limited. The use of viral vectors for delivery of CRISPR/Cas components has recently emerged as a powerful alternative but it requires further exploration. In this review, we discuss the different strategies, principles, applications, and future directions of transgene-free genome editing methods.

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