Recent advances in DNA-free editing and precise base editing in plants

2017 ◽  
Vol 1 (2) ◽  
pp. 161-168 ◽  
Author(s):  
Yi Zhang ◽  
Caixia Gao
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Point Mutations ◽  
Plant Genome ◽  
The Novel ◽  
Future Perspectives ◽  
Delivery Methods ◽  
Base Editing ◽  
Short Palindromic Repeat ◽  
Target Effects

Genome-editing technologies based on the CRISPR (clustered regularly interspaced short palindromic repeat) system have been widely used in plants to investigate gene function and improve crop traits. The recently developed DNA-free delivery methods and precise base-editing systems provide new opportunities for plant genome engineering. In this review, we describe the novel DNA-free genome-editing methods in plants. These methods reduce off-target effects and may alleviate regulatory concern about genetically modified plants. We also review applications of base-editing systems, which are highly effective in generating point mutations and are of great value for introducing agronomically valuable traits. Future perspectives for DNA-free editing and base editing are also 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.

Genome editing of maize

2021 ◽  
pp. 341-376
Author(s):  
Jacob D. Zobrist ◽  
◽  
Morgan McCaw ◽  
Minjeong Kang ◽  
Alan L. Eggenberger ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Target Genes ◽  
Zinc Finger Nucleases ◽  
Maize Genome ◽  
Delivery Methods ◽  
Agricultural Improvement ◽  
Modern Maize ◽  
Associated Proteins ◽  
Short Palindromic Repeat

Developed over thousands of years largely through human intervention, the modern maize genome can now be precisely modified for agricultural improvement and scientific research. This chapter focuses on progress made in recent decades utilizing site-specific nuclease (SSN) technologies in maize genome engineering. Many SSNs, such as meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated proteins (Cas) have been used in maize for both functional analysis and trait improvement. The chapter summarizes the recent innovations related to maize genome editing using SSN technologies, the type of approaches, target genes and traits, and reagent delivery methods. It also discusses the current challenges as well as potential improvements for maize genome engineering protocols.

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.

scholarly journals Exosome/Liposome-like Nanoparticles: New Carriers for CRISPR Genome Editing in Plants

2021 ◽  
Vol 22 (14) ◽  
pp. 7456
Author(s):  
Mousa A. Alghuthaymi ◽  
Aftab Ahmad ◽  
Zulqurnain Khan ◽  
Sultan Habibullah Khan ◽  
Farah K. Ahmed ◽  
...  
Keyword(s):  
Genome Editing ◽  
Viral Vectors ◽  
Polymeric Materials ◽  
Inorganic Nanoparticles ◽  
Plant Genome ◽  
Delivery Methods ◽  
Detailed Consideration ◽  
Crispr System ◽  
Efficient Delivery ◽  
Low Cytotoxicity

Rapid developments in the field of plant genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems necessitate more detailed consideration of the delivery of the CRISPR system into plants. Successful and safe editing of plant genomes is partly based on efficient delivery of the CRISPR system. Along with the use of plasmids and viral vectors as cargo material for genome editing, non-viral vectors have also been considered for delivery purposes. These non-viral vectors can be made of a variety of materials, including inorganic nanoparticles, carbon nanotubes, liposomes, and protein- and peptide-based nanoparticles, as well as nanoscale polymeric materials. They have a decreased immune response, an advantage over viral vectors, and offer additional flexibility in their design, allowing them to be functionalized and targeted to specific sites in a biological system with low cytotoxicity. This review is dedicated to describing the delivery methods of CRISPR system into plants with emphasis on the use of non-viral vectors.

scholarly journals Recent advances in CRISPR/Cas9 and applications for wheat functional genomics and breeding

aBIOTECH ◽  
2021 ◽  
Author(s):  
Jun Li ◽  
Yan Li ◽  
Ligeng Ma
Keyword(s):  
Functional Genomics ◽  
Genome Editing ◽  
Functional Annotation ◽  
Genome Engineering ◽  
Agronomic Traits ◽  
Wheat Breeding ◽  
Breeding Programs ◽  
Base Editing ◽  
The World ◽  
World Food Security

AbstractCommon wheat (Triticum aestivum L.) is one of the three major food crops in the world; thus, wheat breeding programs are important for world food security. Characterizing the genes that control important agronomic traits and finding new ways to alter them are necessary to improve wheat breeding. Functional genomics and breeding in polyploid wheat has been greatly accelerated by the advent of several powerful tools, especially CRISPR/Cas9 genome editing technology, which allows multiplex genome engineering. Here, we describe the development of CRISPR/Cas9, which has revolutionized the field of genome editing. In addition, we emphasize technological breakthroughs (e.g., base editing and prime editing) based on CRISPR/Cas9. We also summarize recent applications and advances in the functional annotation and breeding of wheat, and we introduce the production of CRISPR-edited DNA-free wheat. Combined with other achievements, CRISPR and CRISPR-based genome editing will speed progress in wheat biology and promote sustainable agriculture.

scholarly journals BEAR reveals that increased fidelity variants can successfully reduce the mismatch tolerance of adenine but not cytosine base editors

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
András Tálas ◽  
Dorottya A. Simon ◽  
Péter I. Kulcsár ◽  
Éva Varga ◽  
Sarah L. Krausz ◽  
...  
Keyword(s):  
High Throughput ◽  
Genome Engineering ◽  
Sequence Context ◽  
Base Editing ◽  
Target Dna ◽  
Dna Strands ◽  
Deaminase Domain ◽  
Target Effects ◽  
Target Sets

AbstractAdenine and cytosine base editors (ABE, CBE) allow for precision genome engineering. Here, Base Editor Activity Reporter (BEAR), a plasmid-based fluorescent tool is introduced, which can be applied to report on ABE and CBE editing in a virtually unrestricted sequence context or to label base edited cells for enrichment. Using BEAR-enrichment, we increase the yield of base editing performed by nuclease inactive base editors to the level of the nickase versions while maintaining significantly lower indel background. Furthermore, by exploiting the semi-high-throughput potential of BEAR, we examine whether increased fidelity SpCas9 variants can be used to decrease SpCas9-dependent off-target effects of ABE and CBE. Comparing them on the same target sets reveals that CBE remains active on sequences, where increased fidelity mutations and/or mismatches decrease the activity of ABE. Our results suggest that the deaminase domain of ABE is less effective to act on rather transiently separated target DNA strands, than that of CBE explaining its lower mismatch tolerance.

scholarly journals CRISPR-Cas12a (Cpf1): A Versatile Tool in the Plant Genome Editing Tool Box for Agricultural Advancement

2020 ◽  
Author(s):  
Anindya Bandyopadhyay ◽  
Nagesh Kancharla ◽  
vivek javalkote ◽  
santanu dasgupta ◽  
Thomas Brutnell
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Temperature Stability ◽  
Double Strand Breaks ◽  
Plant Genome ◽  
Strand Breaks ◽  
Guide Rna ◽  
Versatile Tool ◽  
Type V ◽  
Global Population

Global population is predicted to approach 10 billion by 2050, an increase of over 2 billion from today. To meet the demands of growing, geographically and socio-economically diversified nations, we need to diversity and expand agricultural production. This expansion of agricultural productivity will need to occur under increasing biotic, and environmental constraints driven by climate change. Clustered regularly interspaced short palindromic repeats-site directed nucleases (CRISPR-SDN) and similar genome editing technologies will likely be key enablers to meet future agricultural needs. While the application of CRISPR-Cas9 mediated genome editing has led the way, the use of CRISPR-Cas12a is also increasing significantly for genome engineering of plants. The popularity of the CRISPR-Cas12a, the type V (class-II) system, is gaining momentum because of its versatility and simplified features. These include the use of a small guide RNA devoid of trans-activating crispr RNA (tracrRNA), targeting of T-rich regions of the genome where Cas9 is not suitable for use, RNA processing capability facilitating simpler multiplexing, and its ability to generate double strand breaks (DSB) with staggered ends. Many monocot and dicot species have been successfully edited using this Cas12a system and further research is ongoing to improve its efficiency in plants, including improving the temperature stability of the Cas12a enzyme, identifying new variants of Cas12a or synthetically producing Cas12a with flexible PAM sequences. In this review we provide a comparative survey of CRISPR-Cas12a and Cas9, and provide a perspective on applications of CRISPR-Cas12 in agriculture.

scholarly journals CRISPR base editors: genome editing without double-stranded breaks

2018 ◽  
Vol 475 (11) ◽  
pp. 1955-1964 ◽  
Author(s):  
Ayman Eid ◽  
Sahar Alshareef ◽  
Magdy M. Mahfouz
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Genetic Diseases ◽  
Great Promise ◽  
Cytidine Deaminases ◽  
Strand Breaks ◽  
Base Editing ◽  
Potential Applications ◽  
Short Palindromic Repeat ◽  
Basic Biology

The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 adaptive immunity system has been harnessed for genome editing applications across eukaryotic species, but major drawbacks, such as the inefficiency of precise base editing and off-target activities, remain. A catalytically inactive Cas9 variant (dead Cas9, dCas9) has been fused to diverse functional domains for targeting genetic and epigenetic modifications, including base editing, to specific DNA sequences. As base editing does not require the generation of double-strand breaks, dCas9 and Cas9 nickase have been used to target deaminase domains to edit specific loci. Adenine and cytidine deaminases convert their respective nucleotides into other DNA bases, thereby offering many possibilities for DNA editing. Such base-editing enzymes hold great promise for applications in basic biology, trait development in crops, and treatment of genetic diseases. Here, we discuss recent advances in precise gene editing using different platforms as well as their potential applications in basic biology and biotechnology.

scholarly journals Base editors: modular tools for the introduction of point mutations in living cells

2019 ◽  
Vol 3 (5) ◽  
pp. 483-491 ◽  
Author(s):  
Mallory Evanoff ◽  
Alexis C. Komor
Keyword(s):  
Synthetic Biology ◽  
Genome Editing ◽  
Genome Engineering ◽  
Point Mutations ◽  
Living Cells ◽  
Single Base ◽  
New Family ◽  
Alternative Techniques ◽  
Cas Proteins ◽  
Modular Nature

Base editors are a new family of programmable genome editing tools that fuse ssDNA (single-stranded DNA) modifying enzymes to catalytically inactive CRISPR-associated (Cas) endonucleases to induce highly efficient single base changes. With dozens of base editors now reported, it is apparent that these tools are highly modular; many combinations of ssDNA modifying enzymes and Cas proteins have resulted in a variety of base editors, each with its own unique properties and potential uses. In this perspective, we describe currently available base editors, highlighting their modular nature and describing the various options available for each component. Furthermore, we briefly discuss applications in synthetic biology and genome engineering where base editors have presented unique advantages over alternative techniques.

scholarly journals The Solanum tuberosum GBSSI gene: a target for assessing gene and base editing in tetraploid potato

2019 ◽  
Author(s):  
Florian Veillet ◽  
Laura Chauvin ◽  
Marie-Paule Kermarrec ◽  
François Sevestre ◽  
Mathilde Merrer ◽  
...  
Keyword(s):  
Solanum Tuberosum ◽  
Genome Editing ◽  
Genome Engineering ◽  
Basic Research ◽  
Loss Of Function ◽  
Dna Breaks ◽  
Nucleotide Substitutions ◽  
Discrete Variation ◽  
Tetraploid Potato ◽  
Base Editing

AbstractGenome editing has recently become a method of choice for basic research and functional genomics, and holds great potential for molecular plant breeding applications. The powerful CRISPR-Cas9 system that typically produces double-strand DNA breaks is mainly used to generate knockout mutants. Recently, the development of base editors has broadened the scope of genome editing, allowing precise and efficient nucleotide substitutions. In this study, we produced mutants in two cultivated elite cultivars of the tetraploid potato (Solanum tuberosum) using stable or transient expression of the CRISPR-Cas9 components to knockout the amylose-producing StGBSSI gene. We set up a rapid, highly sensitive and cost-effective screening strategy based on high-resolution melting analysis followed by direct Sanger sequencing and trace chromatogram analysis. Most mutations consisted of small indels, but unwanted insertions of plasmid DNA were also observed. We successfully created tetra-allelic mutants with impaired amylose biosynthesis, confirming the loss-of-function of the StGBSSI protein. The second main objective of this work was to demonstrate the proof of concept of CRISPR-Cas9 base editing in the tetraploid potato by targeting two loci encoding catalytic motifs of the StGBSSI enzyme. Using a cytidine base editor (CBE), we efficiently and precisely induced DNA substitutions in the KTGGL-encoding locus, leading to discrete variation in the amino acid sequence and generating a loss-of-function allele. The successful application of base editing in the tetraploid potato opens up new avenues for genome engineering in this species.Key MessageThe StGBSSI gene was successfully and precisely edited in the tetraploid potato using gene and base editing strategies, leading to plants with impaired amylose biosynthesis.

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