Progress in precise and predictable genome editing in plants with base editing

2021 ◽  
pp. 123-146
Author(s):  
Sabine Fräbel ◽  
◽  
Shai J. Lawit ◽  
Jingyi Nie ◽  
David G. Schwark ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Genome Editing ◽  
Gene Editing ◽  
In Planta ◽  
Reading Frame ◽  
Base Editing ◽  
Adenine Base

Base editors are gene editing tools that allow targeted nucleic acid conversions, most commonly C>T and A>G, through pairing of deamination domains with impaired nucleases. Multiple deaminase domains and architectures have been demonstrated in planta across a wide array of species, with both cytosine and adenine base editing frequencies being observed at over 80%. The ability of base editors to introduce nucleic acid diversity while maintaining the same reading frame should make them powerful tools for plant genetic editing moving forward.

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.

scholarly journals Current Advancements and Limitations of Gene Editing in Orphan Crops

2021 ◽  
Vol 12 ◽  
Author(s):  
Matthew Venezia ◽  
Kate M. Creasey Krainer
Keyword(s):  
Gene Editing ◽  
Crop Improvement ◽  
Screening Methods ◽  
In Planta ◽  
Base Editing ◽  
Guide Rna ◽  
Orphan Crops ◽  
Associated Proteins ◽  
Improvement Programs ◽  
Novel Alleles

Gene editing provides precise, heritable genome mutagenesis without permanent transgenesis, and has been widely demonstrated and applied in planta. In the past decade, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) has revolutionized the application of gene editing in crops, with mechanistic advances expanding its potential, including prime editing and base editing. To date, CRISPR/Cas has been utilized in over a dozen orphan crops with diverse genetic backgrounds, leading to novel alleles and beneficial phenotypes for breeders, growers, and consumers. In conjunction with the adoption of science-based regulatory practices, there is potential for CRISPR/Cas-mediated gene editing in orphan crop improvement programs to solve a plethora of agricultural problems, especially impacting developing countries. Genome sequencing has progressed, becoming more affordable and applicable to orphan crops. Open-access resources allow for target gene identification and guide RNA (gRNA) design and evaluation, with modular cloning systems and enzyme screening methods providing experimental feasibility. While the genomic and mechanistic limitations are being overcome, crop transformation and regeneration continue to be the bottleneck for gene editing applications. International collaboration between all stakeholders involved in crop improvement is vital to provide equitable access and bridge the scientific gap between the world’s most economically important crops and the most under-researched crops. This review describes the mechanisms and workflow of CRISPR/Cas in planta and addresses the challenges, current applications, and future prospects in orphan crops.

scholarly journals How to increase precision in Xenopus gene editing and base editing experiments

2021 ◽  
Author(s):  
Stuart Smith
Keyword(s):  
Developmental Biology ◽  
Genome Editing ◽  
Gene Mutation ◽  
Gene Editing ◽  
Base Editing ◽  
Aquatic Vertebrate ◽  
Frog Species ◽  
Vertebrate Embryos ◽  
Biology Research

A review focussed on improvements in genome editing methods for developmental biology research using aquatic vertebrate embryos from Xenopus frog species. It is aimed as an introduction for students learning the differences between CRISPR/Cas9-mediated gene mutation versus base editing strategies. The benefits, limitations and efficiencies achieved by the different methods are discussed.

scholarly journals Base editing and prime editing in laboratory animals

2021 ◽  
pp. 002367722199389
Author(s):  
Federico Caso ◽  
Benjamin Davies
Keyword(s):  
Genome Editing ◽  
Laboratory Animal ◽  
Genome Engineering ◽  
Point Mutations ◽  
Animal Research ◽  
Ease Of Use ◽  
Laboratory Animals ◽  
Genomic Change ◽  
Base Editing ◽  
Adenine Base

Genome editing by programmable RNA-dependent Cas endonucleases has revolutionised the field of genome engineering, achieving targeted genomic change at unprecedented efficiencies with considerable application in laboratory animal research. Despite its ease of use and wide application, there remain concerns about the precision of this technology and a number of unpredictable consequences have been reported, mostly resulting from the DNA double-strand break (DSB) that conventional CRISPR editing induces. In order to improve editing precision, several iterations of the technology been developed over the years. Base editing is one of most successful developments, allowing for single base conversions but without the need for a DSB. Cytosine and adenine base editing are now established as reliable methods to achieve precise genome editing in animal research studies. Both cytosine and adenine base editors have been applied successfully to the editing of zygotes, resulting in the generation of animal models. Similarly, both base editors have achieved precise editing of point mutations in somatic cells, facilitating the development of gene therapy approaches. Despite rapid progress in optimising these tools, base editing can address only a subset of possible base conversions within a relatively narrow window and larger genomic manipulations are not possible. The recent development of prime editing, originally defined as a simple ‘search and replace’ editing tool, may help address these limitations and could widen the range of genome manipulations possible. Preliminary reports of prime editing in animals are being published, and this new technology may allow significant advancements for laboratory animal research.

scholarly journals Plant-based biosensors for detecting CRISPR-mediated genome engineering

2021 ◽  
Author(s):  
Guoliang Yuan ◽  
Md Mahmudul Hassan ◽  
Tao Yao ◽  
Haiwei Lu ◽  
Michael Melesse Vergara ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Genome Editing ◽  
Transient Expression ◽  
Gene Editing ◽  
Genome Engineering ◽  
Protoplast Transformation ◽  
Reliable System ◽  
Base Editing ◽  
Detection Systems ◽  
Cas9 Nuclease

CRISPR/Cas has recently emerged as the most reliable system for genome engineering in various species. However, concerns about risks associated with CRISPR/Cas9 technology are increasing on potential unintended DNA changes that might accidentally arise from CRISPR gene editing. Developing a system that can detect and report the presence of active CRIPSR/Cas tools in biological systems is therefore very necessary. Here, we developed the real-time detection systems that can spontaneously indicate CRISPR-Cas tools for genome editing and gene regulation including CRISPR/Cas9 nuclease, base editing, prime editing and CRISPRa in plants. Using the fluorescence-based molecular biosensors, we demonstrated that the activities of CRISPR/Cas9 nuclease, base editing, prime editing and CRIPSRa can be effectively detected in transient expression via protoplast transformation and leaf infiltration (in Arabidopsis, poplar, and tobacco) and stable transformation in Arabidopsis.

scholarly journals Plant viral vectors: Expanding the Possibilities of Precise Gene Editing in Plant Genomes

2021 ◽  
Author(s):  
Stuti Kujur ◽  
Muthappa Senthil-Kumar ◽  
Rahul Kumar
Keyword(s):  
Genome Editing ◽  
Plant Transformation ◽  
Viral Vectors ◽  
Gene Editing ◽  
Genome Engineering ◽  
Plant Cells ◽  
In Planta ◽  
Plant Genomes ◽  
Efficient Gene ◽  
Transformation Methods

Abstract The lack of a highly efficient method for delivering reagents for genome engineering to plant cells remains a bottleneck in achieving efficient gene-editing in plant genomes. A suite of recent reports uncovers the newly emerged roles of viral vectors, which can introduce gene-edits in plants with high mutation frequencies through in planta delivery. Here, we focus on the emerging protocols that utilized different approaches for virus-mediated genome editing in model plants. Testing of these protocols and the newly identified hypercompact Casɸ systems is needed to broaden the scope of genome-editing in most plant species, including crops, with minimized reliance on conventional plant transformation methods in the future.

scholarly journals Aglycosylated antibody-producing mice for aglycosylated antibody-lectin coupled immunoassay for the quantification of tumor markers (ALIQUAT)

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Nan-Ee Lee ◽  
Sun Hee Kim ◽  
Dae-Yeul Yu ◽  
Eui-Jeon Woo ◽  
Myung-Il Kim ◽  
...  
Keyword(s):  
Gene Editing ◽  
Cross Reactivity ◽  
Cancer Diagnostics ◽  
Chemical Methods ◽  
Base Editing ◽  
Quantitative Measurements ◽  
In Vitro Diagnostic ◽  
Adenine Base ◽  
Disease Specific

Abstract Targeting aberrant glycoforms has been validated for in vitro cancer diagnostic development, and several assays are currently in routine clinical use. Because N-glycans in Fc region of antibodies show cross-reactivity with various lectins, high-quality aglycosylated antibodies are exceptionally important for immunoassay platform-based quantitative measurements. Previously, aglycosylated antibody acquisition relied on incomplete, uneconomical and onerous enzymatic and chemical methods. Here, we edited four murine immunoglobulin G genes using adenine base-editing and homology-directed recombination (HDR)-mediated gene editing methods to generate aglycosylated antibody-producing mice. Resulting aglycosylated antibodies showed required analytical performances without compromised protein stability. Thus, this aglycosylated monoclonal antibody-lectin coupled immunoassay for the quantification of tumour markers (ALIQUAT) method can provide a robust, versatile and accessible immunoassay platform to quantify specific glycoforms in precision cancer diagnostics. Moreover, the engineered mice can be used as a host to produce various aglycosylated antibodies in a convenient and robust fashion, thereby expanding in vitro diagnostic development opportunities that utilize glycoforms as a disease-specific biomarkers.

scholarly journals Genome Editing: An Emerging Tool for Plant Breeders

2020 ◽  
Author(s):  
Anand Kumar ◽  
Karansher Sandhu
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Biotic Resistance ◽  
New Technology ◽  
Crop Improvement ◽  
Modern Society ◽  
New Era ◽  
Crop Varieties ◽  
Gene Information ◽  
Adenine Base

Conventional plant breeding has contributed enormously towards feeding the world and has played crucial roles in the development of modern society. The conventional method creates variation by transferring genes between or within the species. In general, these methods are more expensive and takes more time, to overcome these limitations, new technology is required. Genome editing is a powerful tool for biotechnology applications, with the capacity to alter the function of any gene. With the availability of gene information for the majority of the traits, genome editing emerged as a potential to create a new variation with the introduction of any transgene. The important genome editing tools used nowadays are ZFNs, TALEN, Pentatricopeptide repeats protein, adenine base editor, RNA interference, and CRISPR/Cas9. These tools have opened a new era for crop improvement. Due to the complex genetic architecture of most traits, it is challenging to edit genes controlling them. To overcome these challenges, genome editing provides a broader perspective. Among the above-mentioned tools, CRISPR/Cas9 is the most powerful tool for gene editing. These technologies are being used to create abiotic and biotic resistance crop varieties.

scholarly journals Base Editing: The Ever Expanding Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Tool Kit for Precise Genome Editing in Plants

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 466 ◽  
Author(s):  
Mahmuda Binte Monsur ◽  
Gaoneng Shao ◽  
Yusong Lv ◽  
Shakeel Ahmad ◽  
Xiangjin Wei ◽  
...  
Keyword(s):  
Genome Editing ◽  
Target Genes ◽  
Point Mutations ◽  
Base Substitution ◽  
Future Perspectives ◽  
Specific Point ◽  
Target Region ◽  
Base Editing ◽  
Low Levels ◽  
Adenine Base

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9), a newly developed genome-editing tool, has revolutionized animal and plant genetics by facilitating modification of target genes. This simple, convenient base-editing technology was developed to improve the precision of genome editing. Base editors generate precise point mutations by permanent base conversion at a specific point, with very low levels of insertions and deletions. Different plant base editors have been established by fusing various nucleobase deaminases with Cas9, Cas13, or Cas12a (Cpf1), proteins. Adenine base editors can efficiently convert adenine (A) to guanine (G), whereas cytosine base editors can convert cytosine (C) to thymine (T) in the target region. RNA base editors can induce a base substitution of A to inosine (I) or C to uracil (U). In this review, we describe the precision of base editing systems and their revolutionary applications in plant science; we also discuss the limitations and future perspectives of this approach.

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