This document discusses precision breeding techniques in plants, including their objectives, advantages, and challenges. It covers molecular marker-assisted selection, mutagenesis using chemicals or radiation, and newer gene editing tools like CRISPR/Cas9. CRISPR allows more precise genetic modifications than traditional techniques by targeting specific genes. However, increasing the efficiency of the homology directed repair pathway is still an area of research, as non-homologous end joining is the dominant repair pathway in plants. Overall, precision breeding holds promise for accelerating crop improvement but developing methods for high-efficiency homology directed repair remains important.
Speed Breeding is new technology to develop plants or breeding materials within a short possible time without affect seed viability and yield performance.
FERTILITY RESTORATION IN MALE STERILE LINES AND RESTORER DIVERSIFICATION PROG...Rachana Bagudam
1. FERTILITY RESTORATION IN MALE STERILE LINES AND RESTORER DIVERSIFICATION PROGRAMMES.
2. CONVERSION OF AGRONOMICALLY IDEAL GENOTYPES INTO MALE STERILES.
3. GENERATING NEW CYTONUCLEAR INTERACTION SYSTEM FOR DIVERSIFICATION OF MALE STERILES.
To handle complex Traits like Yield, different stress we must do modification in DNA molecular breeding techniques help us to do such changes in DNA to archive the Goals.
Speed Breeding is new technology to develop plants or breeding materials within a short possible time without affect seed viability and yield performance.
FERTILITY RESTORATION IN MALE STERILE LINES AND RESTORER DIVERSIFICATION PROG...Rachana Bagudam
1. FERTILITY RESTORATION IN MALE STERILE LINES AND RESTORER DIVERSIFICATION PROGRAMMES.
2. CONVERSION OF AGRONOMICALLY IDEAL GENOTYPES INTO MALE STERILES.
3. GENERATING NEW CYTONUCLEAR INTERACTION SYSTEM FOR DIVERSIFICATION OF MALE STERILES.
To handle complex Traits like Yield, different stress we must do modification in DNA molecular breeding techniques help us to do such changes in DNA to archive the Goals.
it cover almost all content in cis/intragesis, right from introduction definition, explanation, production of marker free transgenic, intragenic vector construction, regulatory guide lines, current and future status, limitation, advantage over existing technique, swot analysis etc
its very useful for your seminar and presentations. it contain lot of picture, table, figure for your easy understanding
thank you
Mahesh
The presentation was done as part of the course STAT 504 titled Quantitative Genetics in Second Semester of MSc. Agricultural Statistics at Agricultural College, Bapatla under ANGRAU, Andhra Pradesh
A new era of genomics for plant science research has opened due the complete genome sequencing projects of Arabidopsis thaliana and rice. The sequence information available in public database has highlighted the need to develop genome scale reverse genetic strategies for functional analysis (Till et al., 2003). As most of the phenotypes are obscure, the forward genetics can hardly meet the demand of a high throughput and large-scale survey of gene functions. Targeting Induced Local Lesions in Genome TILLING is a general reverse genetic technique that combines chemical mutagenesis with PCR based screening to identity point mutations in regions of interest (McCallum et al., 2000). This strategy works with a mismatch-specific endonuclease to detect induced or natural DNA polymorphisms in genes of interest. A newly developed general reverse genetic strategy helps to locate an allelic series of induced point mutations in genes of interest. It allows the rapid and inexpensive detection of induced point mutations in populations of physically or chemically mutagenized individuals. To create an induced population with the use of physical/chemical mutagens is the first prerequisite for TILLING approach. Most of the plant species are compatible with this technique due to their self-fertilized nature and the seeds produced by these plants can be stored for long periods of time (Borevitz et al., 2003). The seeds are treated with mutagens and raised to harvest M1 plants, which are consequently, self-fertilized to raise the M2 population. DNA extracted from M2 plants is used in mutational screening (Colbert et al., 2001). To avoid mixing of the same mutation only one M2 plant from each M1 is used for DNA extraction (Till et al., 2007). The M3 seeds produce by selfing the M2 progeny can be well preserved for long term storage. Ethyl methane sulfonate (EMS) has been extensively used as a chemical mutagen in TILLING studies in plants to generate mutant populations, although other mutagens can be effective. EMS produces transitional mutations (G/C, A/T) by alkylating G residues which pairs with T instead of the conservative base pairing with C (Nagy et al., 2003). It is a constructive approach for users to attempt a range of chemical mutagens to assess the lethality and sterility on germinal tissue before creating large mutant populations.
Access to large-scale omics datasets i.e. genomics, transcriptomics, proteomics, metabolomics, phenomics, etc. has revolutionized biology and led to the emergence of systems approaches to advance our understanding of biological processes. With decreasing time and cost to generate these datasets, omics data integration has created both exciting opportunities and immense challenges for biologists, computational biologists, biostatisticians and biomathematicians. Genomics, transcriptomics, proteomics, and metabolomics together they help to bring out the best of characters in plants.
An overview of agricultural applications of genome editing: Crop plantsOECD Environment
The presentation gives an overview of genome editing applications in relation to crop plants. The aim is to have a better understanding of the specific features of genome editing in comparison with classical breeding and genetic engineering techniques. It will give an overview of some examples of agricultural applications that may be on or close to the market or under research and development. It will also consider the possibility of foreseeing future applications (e.g. variations in CRISPR/Cas applications, DNA-free application, agricultural pest control), if possible.
it cover almost all content in cis/intragesis, right from introduction definition, explanation, production of marker free transgenic, intragenic vector construction, regulatory guide lines, current and future status, limitation, advantage over existing technique, swot analysis etc
its very useful for your seminar and presentations. it contain lot of picture, table, figure for your easy understanding
thank you
Mahesh
The presentation was done as part of the course STAT 504 titled Quantitative Genetics in Second Semester of MSc. Agricultural Statistics at Agricultural College, Bapatla under ANGRAU, Andhra Pradesh
A new era of genomics for plant science research has opened due the complete genome sequencing projects of Arabidopsis thaliana and rice. The sequence information available in public database has highlighted the need to develop genome scale reverse genetic strategies for functional analysis (Till et al., 2003). As most of the phenotypes are obscure, the forward genetics can hardly meet the demand of a high throughput and large-scale survey of gene functions. Targeting Induced Local Lesions in Genome TILLING is a general reverse genetic technique that combines chemical mutagenesis with PCR based screening to identity point mutations in regions of interest (McCallum et al., 2000). This strategy works with a mismatch-specific endonuclease to detect induced or natural DNA polymorphisms in genes of interest. A newly developed general reverse genetic strategy helps to locate an allelic series of induced point mutations in genes of interest. It allows the rapid and inexpensive detection of induced point mutations in populations of physically or chemically mutagenized individuals. To create an induced population with the use of physical/chemical mutagens is the first prerequisite for TILLING approach. Most of the plant species are compatible with this technique due to their self-fertilized nature and the seeds produced by these plants can be stored for long periods of time (Borevitz et al., 2003). The seeds are treated with mutagens and raised to harvest M1 plants, which are consequently, self-fertilized to raise the M2 population. DNA extracted from M2 plants is used in mutational screening (Colbert et al., 2001). To avoid mixing of the same mutation only one M2 plant from each M1 is used for DNA extraction (Till et al., 2007). The M3 seeds produce by selfing the M2 progeny can be well preserved for long term storage. Ethyl methane sulfonate (EMS) has been extensively used as a chemical mutagen in TILLING studies in plants to generate mutant populations, although other mutagens can be effective. EMS produces transitional mutations (G/C, A/T) by alkylating G residues which pairs with T instead of the conservative base pairing with C (Nagy et al., 2003). It is a constructive approach for users to attempt a range of chemical mutagens to assess the lethality and sterility on germinal tissue before creating large mutant populations.
Access to large-scale omics datasets i.e. genomics, transcriptomics, proteomics, metabolomics, phenomics, etc. has revolutionized biology and led to the emergence of systems approaches to advance our understanding of biological processes. With decreasing time and cost to generate these datasets, omics data integration has created both exciting opportunities and immense challenges for biologists, computational biologists, biostatisticians and biomathematicians. Genomics, transcriptomics, proteomics, and metabolomics together they help to bring out the best of characters in plants.
An overview of agricultural applications of genome editing: Crop plantsOECD Environment
The presentation gives an overview of genome editing applications in relation to crop plants. The aim is to have a better understanding of the specific features of genome editing in comparison with classical breeding and genetic engineering techniques. It will give an overview of some examples of agricultural applications that may be on or close to the market or under research and development. It will also consider the possibility of foreseeing future applications (e.g. variations in CRISPR/Cas applications, DNA-free application, agricultural pest control), if possible.
The CRISPR/Cas9 system has emerged as one of the leading tools for modifying genomes of organisms ranging from E. coli to humans. Additionally, the simple gene targeting mechanism of CRISPR technology has been modified and adapted to other applications that include gene regulation, detection of intercellular trafficking, and pathogen detection. With a wealth of methods for introducing Cas9 and gRNAs into cells, it can be challenging to decide where to start. In this presentation, Dr Adam Clore describes the CRISPR mechanism and some of the most prominent uses for CRISPR, along with methods where IDT technologies can assist scientists in designing, testing, and executing a variety of CRISPR-mediated experiments. For more informaton, visit: http://www.idtdna.com/crispr
CRISPR-Cas9 is a unique technology that enables geneticists and medical researchers to edit parts of the genome by removing, adding or altering sections of the DNA sequence.
It is currently the simplest, most versatile and precise method of genetic manipulation and is therefore causing a buzz in the science world.
Precision Breeding for Climate-Smart Crops - Integrating Genome Editing and B...Sudip Kundu
Climate change poses a serious threat to global food security, demanding innovative solutions. Precision breeding, leveraging the power of genome editing and bioinformatics, presents a promising approach to develop climate-smart crops. This presentation explores the exciting convergence of these technologies, unveiling their potential to unlock resilient and adaptable crops for a changing world.
Key topics covered:
Climate change challenges: Explore the growing threats to agriculture, including rising temperatures, droughts, floods, and pests.
Precision breeding fundamentals: Demystify genome editing techniques like CRISPR and their role in targeted genetic modifications.
Bioinformatics in action: Discover how computational tools analyze vast genetic data, guiding precise interventions in crops.
Developing climate-smart crops: Learn how scientists harness this combined power to breed for traits like heat tolerance, water efficiency, and disease resistance.
Real-world applications: Witness case studies showcasing the development of climate-resilient crops like wheat, rice, and maize.
Future outlook: Discuss the ethical considerations, regulatory frameworks, and potential breakthroughs shaping the future of precision breeding.
Join us on this journey to explore how precision breeding can:
Boost food security in a changing climate
Empower farmers and ensure sustainable agriculture
Shape a brighter future for generations to come
Don't miss this insightful presentation. Share it with your network and engage in the discussion!
#ClimateSmartCrops #PrecisionBreeding #GenomeEditing #Bioinformatics #FoodSecurity
Targeted Induced Local Lesions IN Genome. Mutations (Single base pair substitution) are created by traditionally used chemical mutagens. Identify SNPs and / or INDELS in a gene / genes of interest from a mutagenized population.
Need to revolutionize the crop breedingamoldchokhat
Mankind is facing an enormous challenge of food insecurity. By the year 2050, the population of the world is projected to increase by 35% to a whopping 9 billion; and an almost doubling of global food production is needed to feed the planet. This additional food for humans and farm animals has to come from the existing land, through maximization of genetic potential and relatively quickly. This demand for unprecedented productivity in agriculture needs to be realized in the presence of growing challenges of climate change, shortsighted land-use practices and increasing cost of agriculture despite the rate of improved adoption of technology in crop breeding. Recent advances in our understanding of genes and genomes combined with development of novel tools in biotechnology will play a vital role in accelerating efforts in plant breeding. Genomics assisted breeding assists the breeders in precise selection to enhance the effectivity and enhancement of the precise selection to develop a new cultivars.
hematic appreciation test is a psychological assessment tool used to measure an individual's appreciation and understanding of specific themes or topics. This test helps to evaluate an individual's ability to connect different ideas and concepts within a given theme, as well as their overall comprehension and interpretation skills. The results of the test can provide valuable insights into an individual's cognitive abilities, creativity, and critical thinking skills
ESR spectroscopy in liquid food and beverages.pptxPRIYANKA PATEL
With increasing population, people need to rely on packaged food stuffs. Packaging of food materials requires the preservation of food. There are various methods for the treatment of food to preserve them and irradiation treatment of food is one of them. It is the most common and the most harmless method for the food preservation as it does not alter the necessary micronutrients of food materials. Although irradiated food doesn’t cause any harm to the human health but still the quality assessment of food is required to provide consumers with necessary information about the food. ESR spectroscopy is the most sophisticated way to investigate the quality of the food and the free radicals induced during the processing of the food. ESR spin trapping technique is useful for the detection of highly unstable radicals in the food. The antioxidant capability of liquid food and beverages in mainly performed by spin trapping technique.
Travis Hills' Endeavors in Minnesota: Fostering Environmental and Economic Pr...Travis Hills MN
Travis Hills of Minnesota developed a method to convert waste into high-value dry fertilizer, significantly enriching soil quality. By providing farmers with a valuable resource derived from waste, Travis Hills helps enhance farm profitability while promoting environmental stewardship. Travis Hills' sustainable practices lead to cost savings and increased revenue for farmers by improving resource efficiency and reducing waste.
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
What are greenhouse gasses how they affect the earth and its environment what is the future of the environment and earth how the weather and the climate effects.
DERIVATION OF MODIFIED BERNOULLI EQUATION WITH VISCOUS EFFECTS AND TERMINAL V...Wasswaderrick3
In this book, we use conservation of energy techniques on a fluid element to derive the Modified Bernoulli equation of flow with viscous or friction effects. We derive the general equation of flow/ velocity and then from this we derive the Pouiselle flow equation, the transition flow equation and the turbulent flow equation. In the situations where there are no viscous effects , the equation reduces to the Bernoulli equation. From experimental results, we are able to include other terms in the Bernoulli equation. We also look at cases where pressure gradients exist. We use the Modified Bernoulli equation to derive equations of flow rate for pipes of different cross sectional areas connected together. We also extend our techniques of energy conservation to a sphere falling in a viscous medium under the effect of gravity. We demonstrate Stokes equation of terminal velocity and turbulent flow equation. We look at a way of calculating the time taken for a body to fall in a viscous medium. We also look at the general equation of terminal velocity.
Remote Sensing and Computational, Evolutionary, Supercomputing, and Intellige...University of Maribor
Slides from talk:
Aleš Zamuda: Remote Sensing and Computational, Evolutionary, Supercomputing, and Intelligent Systems.
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Inter-Society Networking Panel GRSS/MTT-S/CIS Panel Session: Promoting Connection and Cooperation
https://www.etran.rs/2024/en/home-english/
The ability to recreate computational results with minimal effort and actionable metrics provides a solid foundation for scientific research and software development. When people can replicate an analysis at the touch of a button using open-source software, open data, and methods to assess and compare proposals, it significantly eases verification of results, engagement with a diverse range of contributors, and progress. However, we have yet to fully achieve this; there are still many sociotechnical frictions.
Inspired by David Donoho's vision, this talk aims to revisit the three crucial pillars of frictionless reproducibility (data sharing, code sharing, and competitive challenges) with the perspective of deep software variability.
Our observation is that multiple layers — hardware, operating systems, third-party libraries, software versions, input data, compile-time options, and parameters — are subject to variability that exacerbates frictions but is also essential for achieving robust, generalizable results and fostering innovation. I will first review the literature, providing evidence of how the complex variability interactions across these layers affect qualitative and quantitative software properties, thereby complicating the reproduction and replication of scientific studies in various fields.
I will then present some software engineering and AI techniques that can support the strategic exploration of variability spaces. These include the use of abstractions and models (e.g., feature models), sampling strategies (e.g., uniform, random), cost-effective measurements (e.g., incremental build of software configurations), and dimensionality reduction methods (e.g., transfer learning, feature selection, software debloating).
I will finally argue that deep variability is both the problem and solution of frictionless reproducibility, calling the software science community to develop new methods and tools to manage variability and foster reproducibility in software systems.
Exposé invité Journées Nationales du GDR GPL 2024
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
Salas, V. (2024) "John of St. Thomas (Poinsot) on the Science of Sacred Theol...Studia Poinsotiana
I Introduction
II Subalternation and Theology
III Theology and Dogmatic Declarations
IV The Mixed Principles of Theology
V Virtual Revelation: The Unity of Theology
VI Theology as a Natural Science
VII Theology’s Certitude
VIII Conclusion
Notes
Bibliography
All the contents are fully attributable to the author, Doctor Victor Salas. Should you wish to get this text republished, get in touch with the author or the editorial committee of the Studia Poinsotiana. Insofar as possible, we will be happy to broker your contact.
Phenomics assisted breeding in crop improvementIshaGoswami9
As the population is increasing and will reach about 9 billion upto 2050. Also due to climate change, it is difficult to meet the food requirement of such a large population. Facing the challenges presented by resource shortages, climate
change, and increasing global population, crop yield and quality need to be improved in a sustainable way over the coming decades. Genetic improvement by breeding is the best way to increase crop productivity. With the rapid progression of functional
genomics, an increasing number of crop genomes have been sequenced and dozens of genes influencing key agronomic traits have been identified. However, current genome sequence information has not been adequately exploited for understanding
the complex characteristics of multiple gene, owing to a lack of crop phenotypic data. Efficient, automatic, and accurate technologies and platforms that can capture phenotypic data that can
be linked to genomics information for crop improvement at all growth stages have become as important as genotyping. Thus,
high-throughput phenotyping has become the major bottleneck restricting crop breeding. Plant phenomics has been defined as the high-throughput, accurate acquisition and analysis of multi-dimensional phenotypes
during crop growing stages at the organism level, including the cell, tissue, organ, individual plant, plot, and field levels. With the rapid development of novel sensors, imaging technology,
and analysis methods, numerous infrastructure platforms have been developed for phenotyping.
This presentation explores a brief idea about the structural and functional attributes of nucleotides, the structure and function of genetic materials along with the impact of UV rays and pH upon them.
4. Objectives of Plant Breeding
31-08-2021 Darshana Patra 4
Prime objective is to increase crop yield and improve quality of crop produce
https:// www.agronomy.org/science-news/understanding-genetic-basis-drought-tolerant-crops/Biotech info center
Until recently, our ability to generate allelic diversity in plants was limited to
introduction of variants from domesticated and wild species by breeding via
uncontrolled recombination or the use of chemical and physical mutagens—
processes that are lengthy and costly or lack specificity.
5. Farm To Fork Strategy
• for a fair, healthy and environmentally friendly food system
• new innovative genomic techniques accelerate the development of
bio based products
• may play a role in increasing sustainability along the food supply
chain
• provided they are safe for consumers and the environment while
bringing benefits to society as a whole.
• accelerate the process of reducing dependency of pesticides
31-08-2021 Darshana Patra 5
6. Molecular Marker Assisted Selection Breeding
31-08-2021 Darshana Patra 6
MAS refers to the use of DNA markers that are tightly-linked to target loci
Assumption: DNA markers can reliably predict phenotype
7. MAS Breeding
31-08-2021 Darshana Patra 7
Pros :
• Similar to traditional breeding , not regulated
• Accelerating breeding process
• Easier for stacking multiple traits within the same
cultivar
Cons:
• Must know genomic and genetic background
• Very costly
• False markers
8. Don’t underestimate the power of genetic variability
Classical Breeding by selecting stem, lateral bud, terminal bud, flower cluster, stem & flower, leaf
31-08-2021 Darshana Patra Discover biology 3/e fig 16-9 W WMNorton & Com Inc 8
12. Pros and Cons of Mutation Breeding
31-08-2021 Darshana Patra 12
Pros:
• Induction of desirable mutant which is absent in natural plant materials
• Not regulated ecologically, environmentally friendly
• Straightforward phenotypic selection, technically easy
Cons:
• Generally random and unpredictable
• Good mutations come with bad mutations
• Need large mutant pool to identify good one
• Costly and slow
13. Enhancing genetic variability with
mutagens
• Used in the last 70 years for breeding.
• More than 3000 cultivars worldwide
• Many further unwanted (off-site) mutations
31-08-2021 Darshana Patra 13
14. High Precision : no or less non-targeted mutations
Traditional Mutagenesis Vs Targeted Mutagenesis
31-08-2021 Darshana Patra 14
15. Going “Bio” : Enzymes can do it better
Site-specific induction of DSBs : Natural inheritance is
governed by molecular scissors
31-08-2021 Darshana Patra 15
16. Plant GE Toolbox: Edit Delete Move
• Gene editing provides a faster and precise way to create new variation,
dominated by the creation of short insertion and deletion mutations leading
to loss of gene function, due to the dependence of editing outcomes on DNA
repair pathway choices intrinsic to higher eukaryotes.
• Other types of edits such as point mutations and precise and pre-designed
targeted sequence insertions have rarely been implemented , despite
providing means to modulate the expression of target genes or to engineer
the function and stability of their protein products.
31-08-2021 Darshana Patra 16
17. Designer Plants
• Custom editing by regulation of repair pathway choices or by
taking advantage of alternative types of DNA repair
• The advent of novel gene editing tools are independent of
DNA double-strand break repair, and methods completely
independent of host DNA repair processes
31-08-2021 Darshana Patra 17
18. Precision Breeding
• A plant breeding approach in which a phenotypic trait of
interest is selected by means of identifying a functional
marker that is directly derived from the genomic region of a
trait-controlling gene
• Selections are based on the polymorphic genic regions linked
with a trait of interest
• Availability of genomic resources are of utmost importance
for making FM
31-08-2021 Darshana Patra 18
19. The next step : Breeding at the speed of light
31-08-2021 Darshana Patra 19
20. CRISPR Loci induce acquired
immunity in bacteria against the virus
infection or plasmid transfer
31-08-2021 Darshana Patra 20
21. 3 Options : Precise edits with CRISPR
• Make use of host cell HDR
– Involve a DSB.
– Capable of very large insertions
• Use enzymatic base editing
– No DSB repair. Single base edits typically
• Use prime editing
– No DSB repair. Capable of all edits <80 bases
31-08-2021 Darshana Patra 21
22. Gene editing via CRISPR/Cas
• CRISPR/Cas is more efficient and cheaper
• Directed , off-site mutations can be avoided
• Any gene can be targeted
• A row of improved traits introduced in different crops already
31-08-2021 Darshana Patra 22
Steps
• Binding with NGG
PAM (spcas9)
• R loop = ds to ss DNA
• sgRNA binding with
homologous
sequence
• Cas9 makes a cut
23. Features of CRISPR/Cas9 GE
31-08-2021 Darshana Patra 23
• High precision, high efficiency with unprecedented ability to generate targeted
and specific mutations
• Procedures are identical to genetic modification
• Final products are similar to traditional breeding
• Deactivating one or multiple genes, up/down regulating genes, early pathogen
detection
• Precise modifications using base editors, prime editing and HDR
• Targeted insertion of transgene
25. Genomic double- strand break (DSB) generation is followed by different cellular repair pathways. Error-prone non-homologous
end joining (NHEJ) and microhomology-mediated end joining (MMEJ)pathways create the majority of mutations throughout the
cell cycle. Homology directed repair (HDR), active in S/G2 phases of the cell cycle, repairs DSB without error.
CELLULAR REPAIR PATHWAY
31-08-2021 Darshana Patra 25
28. 31-08-2021 Darshana Patra Chen et al 2019 28
Staggered cuts by SpCas9
– HNH cleaves target strand at - 3 position
– RuvC can make a cut at either -3, -4, -5, or even further
29. Hypothetical model
31-08-2021 Darshana Patra 29
Staggered cuts by SpCas9
• HNH cleaves target strand at - 3
position
• RuvC can make a cut at either
-3, -4,-5, or even further
Generation of 1 bp insertion during
CRISPR/Cas9-induced DSB repair
30. Predicting precise edited products
31-08-2021 Darshana Patra Molla et al 2019 30
Prediction of CRISPR/Cas9-induced
mutations
31. Scientific risk assesment of gene
edited plants
• DSB repair is a natural process, mutations occur spontaneously all of the time
• Plants with CRISPR/Cas-induced or spontaneous mutations cannot be discriminated and
are nature-identical
• Classical mutagenized plants are exempted from regulation due to a ‘long safety record ‘
• Edited Plants are at least as safe as mutagenized crops
31-08-2021 Darshana Patra 31
32. Out of total cellular repair events..
31-08-2021 Darshana Patra 32
33. Precision editing by HDR
31-08-2021 Darshana Patra 33
• Coincident error prone NHEJ repair confounds HDR strategies
• Yields a majority of random mutants and a minority of accurate HDR
corrections
• NHEJ occurs in G1/S while HDR occurs in G2/M
• A Cas9-geminin fusion imparts G2/M expression on Cas9
• Biased towards HDR events
34. 31-08-2021 Darshana Patra 34
Published : 8 July 2021
• Here, fast, efficient and reproducible targeted nucleotide substitutions in sugarcane
reported, enabling precise co-editing of multiple alleles via template-mediated and
homology-directed repair (HDR) of DNA double strand breaks induced by the
programmable nuclease CRISPR/Cas9
• Selected gene variants into elite cultivars without crossing and associated linkage
drag.
35. Precise editing by HDR mediated allele
replacement (gene targeting)
• In the GT approach, a DRT is constructed by flanking the desired
sequence modifications on each side by regions of homology to the
target locus, often referred to as homology arms.
• When the DRT is delivered to a target cell and a DSB is
simultaneously induced in the genome, the DRT can be used for
HDR that proceeds via synthesis-dependent strand annealing (HDR
repair in plants see Knoll et al. 2014), resulting in incorporation of
the edits in the genome.
31-08-2021 Darshana Patra 35
36. Low HDR frequency in somatic plant cells
31-08-2021 Darshana Patra 36
37. Multi allelic precision ns conferred
herbicide tolerance
• Frequency of HDR in somatic cells is negligible, making routine allele replacement at scale in
plants impractical.
• In fact, GT is usually successful in only 0.1–1% of recovered plants.
• Although some of the improvements described below have increased these efficiencies,
surpassing the 10% mark can be considered exceptional.
• In stark contrast, NHEJ repair of targeted DSBs can generate indels with close to 100%
efficiency in some species (Pan et al. 2016; Ueta et al.2017; Lee et al. 2019b; Malzahn et al.
2019).
• This difference is partly due to the restriction of HDR to the late S and G2/M phases of cell
cycle, as opposed to NHEJ which operates in both dividing and non- dividing cells (Mao et al.
2008; Charbonnel et al. 2011).
• Simply put, the main hurdle to effective GT is the need to create conditions under which HDR
is favored over NHEJ.
31-08-2021 Darshana Patra 37
38. Strategies to improve HDR mediated genome editing
Manipulation
of DNA repair
pathway
Manipulation
of donor
template
Adding new
functions to
Cas enzymes
31-08-2021 Darshana Patra 38
Combinatorial approach
40. Anti Nutritional Factor : Gliadin
31-08-2021 Darshana Patra 40
Ingestion of antigenic
compound i.e. foreign
body Gliadin develop auto
immune response, destroy
the vili (absorb nutrients
from food)structure of
intestine
41. Target epitope as antigenic : MALDI-TOF analysis of the gliadin extracts from T545 , T553 and the BW208 wild type.
PBJ : Susana Sanchez-Leon et. al {18 September 2017}
31-08-2021 Darshana Patra 41
42. Wild species that are heat
and salt stress resistant
can be transformed into
novel crops
31-08-2021 Darshana Patra 42
43. Genetic Engineering
31-08-2021 Darshana Patra 43
Pros :
• Fast way to verify gene function
• Precisely modify crop productivity and quality
Cons :
• Necessary to know gene function
• Very costly, complicated procedures
• Heavily regulated
44. Complimentary to Traditional
Breeding : Solution to Linkage Drag
31-08-2021 Darshana Patra 44
Linkage Drag - Nightmare for Traditional Plant Breeders
Gene editing can remove linkage drag to create better plants
Never has progenies with good
flavor and better postharvest
quality,diseaseresistant tomato
Breaking genetic linkages by CRISPR/Cas
45. Crop Breeding : towards precision
Genome editing: improving a trait by precisely modifying the target genes or regulatory elements
or rearranging chromosomes in elite varieties.
31-08-2021 Darshana Patra Chen et al 2019 45
46. What makes it Different?
31-08-2021 Darshana Patra 46
47. CRISPR Base editing without DNA cleavage
31-08-2021 Darshana Patra 47
• Recruit catalytic domains of DNA deaminases
• Use D10A nickase to nick non-edited strand
to stimulate repair
• No DSB
• Nick repair is very accurate and fast
• Can target most disease associated SNPs
48. Prime editing - CRISPR without DNA breaks
31-08-2021 Darshana Patra 48
49. Prime Editing - CRISPR without DSB
31-08-2021 Darshana Patra 49
• Does not rely on host cell DSB repair to make an edit
• A Cas9 nickase is used to locate and prime the editing
• The intended edit is added to the guide RNA template
• A fused reverse transcriptase incorporates the edit
• Host cell repair incorporates the edit
55. Application of GE Techniques for Crop
Improvement
31-08-2021 Darshana Patra 55
56. Most efficient allele replacement methods for
major plant model and crop species
31-08-2021 Darshana Patra 56
The choice of BE for each application in order to avoid potentialbystander mutationsand other editing byproducts
should be carefullyconsideredbased on :
• the availabilityof PAM sequence properlypositioned in the target sequence
• size of the editing window
• specificity
Citationsand application of selection refer to the highestachieved editing efficiencyfor the given approach and species.
*Direct selection for the gene editing event, DRT donor repair template
62. Take home message
31-08-2021 Darshana Patra 62
• Genome editing requires similar procedures used for Genetic
Engineering (GMOs), yet creates precise mutation in plant genomes
containing non-foreign DNA
• Resulting products are indistinguishable from products of natural
variability or mutagenesis, yet genome edited plants are regulated
case-by-case
• Limitation of genome editing application are plant transformation
pipeline and genome availability
63. References
• Chen, Kunling, et al. "CRISPR/Cas genome editing and precision plant breeding in
agriculture." Annual review of plant biology 70 (2019): 667-697.
• García-Molina, María Dolores, et al. "Gluten free wheat: are we there?." Nutrients 11.3
(2019): 487.
• Oz, Mehmet Tufan, et al. "CRISPR/Cas9-Mediated Multi-Allelic Gene Targeting in Sugarcane
Confers Herbicide Tolerance." Frontiers in Genome Editing (2021): 15.
• Sedeek, Khalid EM, Ahmed Mahas, and Magdy Mahfouz. "Plant genome engineering for
targeted improvement of crop traits." Frontiers in plant science 10 (2019): 114
31-08-2021 Darshana Patra 63