A concise and well fabricated presentation the current techniques used for plant genome editing including CRISPER/cas9 system, TALENS, TELES, ZINC FINGER NUCLEASES(ZFN), HEJ (homologous endjoing) and many other high throughout techniques along references.
Genome editing is one of the most important tools which supports genetic engineering. It is based on the naturally occurring mechanism of DNA recombination which involves the initiation of breaks with the double stranded DNA followed by repair by the endogenous DNA polymerases.
Conventional techniques such as gene knockouts using P-elements and transposable genetic elements have been superseded by more accurate genome editing methods such as TALENs and CRISPR/Cas.
RNA interference (RNAi): Cellular process by which an mRNA is targeted for degradation by a dsRNA with a strand complementary to a fragment of such mRNA.
A concise and well fabricated presentation the current techniques used for plant genome editing including CRISPER/cas9 system, TALENS, TELES, ZINC FINGER NUCLEASES(ZFN), HEJ (homologous endjoing) and many other high throughout techniques along references.
Genome editing is one of the most important tools which supports genetic engineering. It is based on the naturally occurring mechanism of DNA recombination which involves the initiation of breaks with the double stranded DNA followed by repair by the endogenous DNA polymerases.
Conventional techniques such as gene knockouts using P-elements and transposable genetic elements have been superseded by more accurate genome editing methods such as TALENs and CRISPR/Cas.
RNA interference (RNAi): Cellular process by which an mRNA is targeted for degradation by a dsRNA with a strand complementary to a fragment of such mRNA.
Transcriptomics is the study of RNA, single-stranded nucleic acid, which was not separated from the DNA world until the central dogma was formulated by Francis Crick in 1958, i.e., the idea that genetic information is transcribed from DNA to RNA and then translated from RNA into protein.
Transgene-free CRISPR/Cas9 genome-editing methods in plantsCIAT
"Transgene-free CRISPR/Cas9 genome-editing methods in plants" by Matthew R. Willmann, Ph.D. Director, Plant Transformation Facility College of Agriculture and Life Sciences, School of Integrative Plant Science, Cornell University.
Genotyping by Sequencing is a robust,fast and cheap approach for high throughput marker discovery.It has applications in crop improvement programs by enhancing identification of superior genotypes.
Banoth Madhu: Map based gene cloning in plant. In the process of map-based cloning, one starts with a mutant and eventually identifies the gene responsible for the altered phenotype, allowing the plant to tell you what genes are important in the physiological process of interest and using the genetic relationship between a gene and a marker as the basis for beginning a search for a gene
An insight into the reverse genetics in fisheries research. it includes a brief history about the reverse genetics, background, techniques applied, recovery of virus and zebrafish research
Transcriptomics is the study of RNA, single-stranded nucleic acid, which was not separated from the DNA world until the central dogma was formulated by Francis Crick in 1958, i.e., the idea that genetic information is transcribed from DNA to RNA and then translated from RNA into protein.
Transgene-free CRISPR/Cas9 genome-editing methods in plantsCIAT
"Transgene-free CRISPR/Cas9 genome-editing methods in plants" by Matthew R. Willmann, Ph.D. Director, Plant Transformation Facility College of Agriculture and Life Sciences, School of Integrative Plant Science, Cornell University.
Genotyping by Sequencing is a robust,fast and cheap approach for high throughput marker discovery.It has applications in crop improvement programs by enhancing identification of superior genotypes.
Banoth Madhu: Map based gene cloning in plant. In the process of map-based cloning, one starts with a mutant and eventually identifies the gene responsible for the altered phenotype, allowing the plant to tell you what genes are important in the physiological process of interest and using the genetic relationship between a gene and a marker as the basis for beginning a search for a gene
An insight into the reverse genetics in fisheries research. it includes a brief history about the reverse genetics, background, techniques applied, recovery of virus and zebrafish research
Functional genomics is a general approach toward understanding how the genes of an organism work together by assigning new functions to unknown genes. Information about the hypothesized function of an unknown gene may be deduced from its sequence structure using already known functions of similar genes as the basis for comparison. Gene function analysis therefore necessitates the analysis of temporal and spatial gene expression patterns (Yunbi Xu et al , Plant Molecular Biology (2005) ).
A powerful non-transgenic reverse genetics method that combines chemical mutagenesis with PCR based screening to identify point mutations in regions of interest.
EcoTILLING is a molecular technique that is similar to TILLING, except that its objective is to uncover natural genetic variation as opposed to induced mutations.
Genetic Transformation of Maize: Conventional Methods and Precision Geno...Ananya Sinha
Genetic Transformation of Maize: Conventional Methods and Precision Genome Modification
This is a summary of the above chapter mentioned in "Biotechnology of Major Cereals".
The precision genome modification is defined majorly focused on comparing the conventional genome modification methods with the modern ones.
The basic food law is intended to assure consumers that foods are pure and wholesome, safe to eat, and produced under sanitary conditions. Generally, food law prohibits importation and distribution of food products that are adulterated, or have labels that are false or misleading in any context.
Soil and water conditions. ...
Keep an eye on the forecast for heavy rainfall events. ...
Calibrate, inspect, and maintain manure application equipment. ...
Separation distances for land application. ...
Irrigation of manure sources. ...
Savvy stockpiling and dry manure management.
Based on the mode of action, the major food preservation techniques can be categorized as: (1) slowing down or inhibiting chemical deterioration and microbial growth, (2) directly inactivating bacteria, yeasts, molds, or enzymes, and (3) avoiding recontamination before and after processing.
Food processing waste is derived from the processing of biological materials and is, in the main, biodegradable. Biowaste is defined in the landfill directive as 'waste capable of undergoing anaerobic or aerobic decomposition such as food and garden waste, and paper and cardboard
Water has a wide variety of uses in food production, for cleaning, sanitation, and manufacturing purposes. In addition to being an ingredient in many foods, it may be used for various other operations, such as for growing, unloading, fluming, washing, brining, ice manufacture, and in sanitation and in hygiene programs.
The environmental damage of food production from conventional agriculture is not limited to deforestation and pollutants associated with crop growth. Harvesting the crop represents a significant amount of nutrients, water, and energy being taken from the land.
The basis for sanitation is the removal of soils from the manufacturing environment. There are many benefits to this process. From a food safety standpoint, there is the removal of pathogenic organisms, prevention of the formation of biofilms and removal of potentially harmful chemicals from food contact surfaces.
Food packaging is defined as enclosing food to protect it from tampering or contamination from physical, chemical, and biological sources, with active packaging being the most common packaging system used for preserving food products.
Sugar, salt, nitrites, butylated hydroxy anisol (BHA), butylated hydroxyl toluene (BHT), tert-butylhydroquinone (TBHQ), vinegar, citric acid, and calcium propionate are all chemicals that preserve foods. Salt, sodium nitrite, spices, vinegar, and alcohol have been used to preserve foods for centuries.
Sugaring is a food preservation method similar to pickling. Sugaring is the process of desiccating a food by first dehydrating it, then packing it with pure sugar. This sugar can be crystalline in the form of table or raw sugar, or it can be a high sugar density liquid such as honey, syrup or molasses.
Removing the moisture from food helps prevent bacterial and fungal growth which would ruin stored foods. Smoking is a method of drying that also imparts flavor to the food (usually meat items), and smoke helps keep bacteria-carrying-insects away during the drying process.
Microwave penetrates inside the food materials resulting in entire internal cooking of whole volume of food rapidly and uniformly reducing the processing time and energy. This fast heat transfer in turn results in preservation of nutrients, vitamins contents, flavor, sensory characteristics, and color of food
Food irradiation (the application of ionizing radiation to food) is a technology that improves the safety and extends the shelf life of foods by reducing or eliminating microorganisms and insects. Like pasteurizing milk and canning fruits and vegetables, irradiation can make food safer for the consumer
Food irradiation is the process of exposing food and food packaging to ionizing radiation, such as from gamma rays, x-rays, or electron beams. Wikipedia
Low dose (up to 1 kGy): Inhibit sprouting (potatoes, onions, yams, garlic)
Lowering the temperature of food so that microbes and enzymes are inactivated.
Moisture is changed to ice and microbes become inactive without water.
Packaging food maintains the colour, flavour and texture.
Fast freezing (-25ºC) helps maintain nutritive value and texture of food.
Chilling is an important activity in food processing. Foods are chilled to extend shelf life by reducing biochemical reactions and microbial activity. Temperature control is essential in order to prevent spoilage and food safety concerns during storage.1
Drying is a mass transfer process consisting of the removal of water or another solvent by evaporation from a solid, semi-solid or liquid. This process is often used as a final production step before selling or packaging products.
Food drying is a method of food preservation in which food is dried (dehydrated or desiccated). Drying inhibits the growth of bacteria, yeasts, and mold through the removal of water.
Dehydration has been used widely for this purpose since ancient times; the earliest known practice is 12,000 B.C. by inhabitants of the modern Middle East and Asia regions. Drying is a simple method for preserving food.
Dried foods make great healthy and tasty snacks. They are good for lunches, travel, backpacking, hiking, and camping plus many other activities. Most types of foods can be dried. Drying is an ancient method of food preservation.
Most foods will not support the growth of bacteria if their water activity is less than 0.85, because at this water activity there is not enough water available for the bacteria to grow.
However, yeasts can grow at water activities as low as 0.70, while some molds will grow even at water activities as low as 0.60!
Foods with water activities in this range usually have preservatives added to prevent the growth of yeasts and molds.
Acidic foods with a pH less than 4.6, such as tomato sauce, retard the growth of microorganisms. Thus an acidic food with a water activity less than 0.85 is relatively shelf stable, especially if it is stored in the refrigerator.
In this case, low pH, water activity and temperature combine to provide good insurance against the growth of harmful pathogens.
Sorption is a physical and chemical process by which one substance becomes attached to another.
Sorption includes both adsorption & absorption
e.g., liquids being absorbed by a solid or gases being absorbed by a liquid, cotton dipped in ink.
Sorption the process in which one substance takes up or holds another; adsorption or absorption
Sorption is a process in which a solute moves from a fluid to a particulate solid.
The food sorption isotherm describes the thermodynamic relationship between water activity and the equilibrium of the moisture content of a food product at constant temperature and pressure. ...
The typical shape of an isotherm reflects the way in which the water binds the system.
Water plays many very important roles in food. It affects texture (dry and brittle versus moist and soft), enables the activity of enzymes and chemical reactions to occur (acts as a solvent), supports the growth of microorganisms, makes it possible for large molecules like polysaccharides and proteins to move about and interact, and conducts heat within food.
Many foods such as meat, poultry, seafood, fruits and vegetables are composed of 75% and more water, so water is the most abundant component in many fresh foods. Other foods such as dairy products, and fresh baked goods also contain high levels of water (about 35% or more). Foods that are high in moisture are at
risk of contamination from the growth of microorganisms such as bacteria, yeast and mold, while dry foods like pasta generally have long shelf lives.
Transcript: Selling digital books in 2024: Insights from industry leaders - T...BookNet Canada
The publishing industry has been selling digital audiobooks and ebooks for over a decade and has found its groove. What’s changed? What has stayed the same? Where do we go from here? Join a group of leading sales peers from across the industry for a conversation about the lessons learned since the popularization of digital books, best practices, digital book supply chain management, and more.
Link to video recording: https://bnctechforum.ca/sessions/selling-digital-books-in-2024-insights-from-industry-leaders/
Presented by BookNet Canada on May 28, 2024, with support from the Department of Canadian Heritage.
Smart TV Buyer Insights Survey 2024 by 91mobiles.pdf91mobiles
91mobiles recently conducted a Smart TV Buyer Insights Survey in which we asked over 3,000 respondents about the TV they own, aspects they look at on a new TV, and their TV buying preferences.
Neuro-symbolic is not enough, we need neuro-*semantic*Frank van Harmelen
Neuro-symbolic (NeSy) AI is on the rise. However, simply machine learning on just any symbolic structure is not sufficient to really harvest the gains of NeSy. These will only be gained when the symbolic structures have an actual semantics. I give an operational definition of semantics as “predictable inference”.
All of this illustrated with link prediction over knowledge graphs, but the argument is general.
UiPath Test Automation using UiPath Test Suite series, part 4DianaGray10
Welcome to UiPath Test Automation using UiPath Test Suite series part 4. In this session, we will cover Test Manager overview along with SAP heatmap.
The UiPath Test Manager overview with SAP heatmap webinar offers a concise yet comprehensive exploration of the role of a Test Manager within SAP environments, coupled with the utilization of heatmaps for effective testing strategies.
Participants will gain insights into the responsibilities, challenges, and best practices associated with test management in SAP projects. Additionally, the webinar delves into the significance of heatmaps as a visual aid for identifying testing priorities, areas of risk, and resource allocation within SAP landscapes. Through this session, attendees can expect to enhance their understanding of test management principles while learning practical approaches to optimize testing processes in SAP environments using heatmap visualization techniques
What will you get from this session?
1. Insights into SAP testing best practices
2. Heatmap utilization for testing
3. Optimization of testing processes
4. Demo
Topics covered:
Execution from the test manager
Orchestrator execution result
Defect reporting
SAP heatmap example with demo
Speaker:
Deepak Rai, Automation Practice Lead, Boundaryless Group and UiPath MVP
DevOps and Testing slides at DASA ConnectKari Kakkonen
My and Rik Marselis slides at 30.5.2024 DASA Connect conference. We discuss about what is testing, then what is agile testing and finally what is Testing in DevOps. Finally we had lovely workshop with the participants trying to find out different ways to think about quality and testing in different parts of the DevOps infinity loop.
JMeter webinar - integration with InfluxDB and GrafanaRTTS
Watch this recorded webinar about real-time monitoring of application performance. See how to integrate Apache JMeter, the open-source leader in performance testing, with InfluxDB, the open-source time-series database, and Grafana, the open-source analytics and visualization application.
In this webinar, we will review the benefits of leveraging InfluxDB and Grafana when executing load tests and demonstrate how these tools are used to visualize performance metrics.
Length: 30 minutes
Session Overview
-------------------------------------------
During this webinar, we will cover the following topics while demonstrating the integrations of JMeter, InfluxDB and Grafana:
- What out-of-the-box solutions are available for real-time monitoring JMeter tests?
- What are the benefits of integrating InfluxDB and Grafana into the load testing stack?
- Which features are provided by Grafana?
- Demonstration of InfluxDB and Grafana using a practice web application
To view the webinar recording, go to:
https://www.rttsweb.com/jmeter-integration-webinar
Kubernetes & AI - Beauty and the Beast !?! @KCD Istanbul 2024Tobias Schneck
As AI technology is pushing into IT I was wondering myself, as an “infrastructure container kubernetes guy”, how get this fancy AI technology get managed from an infrastructure operational view? Is it possible to apply our lovely cloud native principals as well? What benefit’s both technologies could bring to each other?
Let me take this questions and provide you a short journey through existing deployment models and use cases for AI software. On practical examples, we discuss what cloud/on-premise strategy we may need for applying it to our own infrastructure to get it to work from an enterprise perspective. I want to give an overview about infrastructure requirements and technologies, what could be beneficial or limiting your AI use cases in an enterprise environment. An interactive Demo will give you some insides, what approaches I got already working for real.
The Art of the Pitch: WordPress Relationships and SalesLaura Byrne
Clients don’t know what they don’t know. What web solutions are right for them? How does WordPress come into the picture? How do you make sure you understand scope and timeline? What do you do if sometime changes?
All these questions and more will be explored as we talk about matching clients’ needs with what your agency offers without pulling teeth or pulling your hair out. Practical tips, and strategies for successful relationship building that leads to closing the deal.
Accelerate your Kubernetes clusters with Varnish CachingThijs Feryn
A presentation about the usage and availability of Varnish on Kubernetes. This talk explores the capabilities of Varnish caching and shows how to use the Varnish Helm chart to deploy it to Kubernetes.
This presentation was delivered at K8SUG Singapore. See https://feryn.eu/presentations/accelerate-your-kubernetes-clusters-with-varnish-caching-k8sug-singapore-28-2024 for more details.
GraphRAG is All You need? LLM & Knowledge GraphGuy Korland
Guy Korland, CEO and Co-founder of FalkorDB, will review two articles on the integration of language models with knowledge graphs.
1. Unifying Large Language Models and Knowledge Graphs: A Roadmap.
https://arxiv.org/abs/2306.08302
2. Microsoft Research's GraphRAG paper and a review paper on various uses of knowledge graphs:
https://www.microsoft.com/en-us/research/blog/graphrag-unlocking-llm-discovery-on-narrative-private-data/
Search and Society: Reimagining Information Access for Radical FuturesBhaskar Mitra
The field of Information retrieval (IR) is currently undergoing a transformative shift, at least partly due to the emerging applications of generative AI to information access. In this talk, we will deliberate on the sociotechnical implications of generative AI for information access. We will argue that there is both a critical necessity and an exciting opportunity for the IR community to re-center our research agendas on societal needs while dismantling the artificial separation between the work on fairness, accountability, transparency, and ethics in IR and the rest of IR research. Instead of adopting a reactionary strategy of trying to mitigate potential social harms from emerging technologies, the community should aim to proactively set the research agenda for the kinds of systems we should build inspired by diverse explicitly stated sociotechnical imaginaries. The sociotechnical imaginaries that underpin the design and development of information access technologies needs to be explicitly articulated, and we need to develop theories of change in context of these diverse perspectives. Our guiding future imaginaries must be informed by other academic fields, such as democratic theory and critical theory, and should be co-developed with social science scholars, legal scholars, civil rights and social justice activists, and artists, among others.
FIDO Alliance Osaka Seminar: Passkeys at Amazon.pdf
Advanced genome & epigenome editing tools.pptx
1. BY
Assist. Prof.
Dr. Berciyal Golda. P
VICAS
Advanced genome & epigenome editing tools
For manupulation of useful microbes/strains&
their applications
2. Introduction
Genome is a fancy word for all your DNA. From potatoes
to puppies, all living organisms have their own genome. Each
genome contains the information needed to build and maintain
that organism throughout its life.
an organism, including a virus. The genome
is the full complement genetic information in a cell,
and contains the programme required for that
cell to function.
3. WhatisAGENOME?
• A genome is the genetic material of an organism. It consists of DNA
(or RNA in RNA viruses). The genome includes both the
genes (the coding regions), the noncoding DNA and the genetic material
of the mitochondria and chloroplasts.
• The term genome was created in 1920 by Hans Winkler, professor
of botany at the University of Hamburg, Germany.
• The genome broadly refers to the total amount of DNA of a single
cell (haploid cell in the case of a diploid organism) of an
organism, including its genes.
“The whole hereditary information of an organism that is
encoded in the DNA”
4. Genes provide the information for making all proteins that are
necessary for the expression of characters.
Characters refers to how an organism looks, its physiology, its
ability to fight infections and even its behavior.
GENE PROTEIN CHARACTER
The genome is found inside every cell, and in those that have
nucleus, the genome is situated inside the nucleus. It is a part of the DNA
molecule.
DNA sequencing techniques enables scientists to determine the exact
order or sequence of the bases of a genome.
5.
6. • The sequence information of the genome will show,
• the position of every gene along the chromosome,
• the regulatory regions that flank each gene &
• the coding sequence that determines the protein produce by each
gene.
WHY SEQUENCE GENOMES?
• Because there is a need to put information about the genomes of flora and
fauna in the context of the fields that they serve.
• Genomic sciences will serve those that choose genetic modification as a
method for crop improvement as well as those that apply conventional
breeding methods to improve and develop agricultural practices.
7. • This information is used by physiologists and scientists in research
determining relationships between stress, genes and yield potential etc.
• It can also be used to produce sufficient amounts of safe and nutritious
food in times of increased population growth.
• It can be used to conserve and protect agricultural and other environments.
• It can serve the farmer/producer under increasing financial pressure by
providing higher yields through improve varieties.
• It can be used to conserve and protect agricultural and other environments.
8. • We need information and technology to
– improve human health,
– harness natural energy,
– understand and react in a positive manner to global climate change,
– clean up our environment and
– ensure food safety.
9. WHAT IS GENOMICS?
• Genomics is the sub discipline of genetics devoted to the
–mapping,
–sequencing ,
–and functional analysis of genomics.
The field includes studies of introgenomic phenomena such as
heterosis, epistasis, pleiotropy and other interactions between loci
and alleles within the genome.
10. How is Genomics different from Genetics?
– Genetics looks at single genes, one at a time, like a picture
or snapshot.
-Genomics looks at the big picture and examines all the
genes as an entire system.
11. GENOMEEDITING
• also includes making alterations to non-coding regions of
genomes and to epigenomes.
• Targeted interventions
• Alter the structural or functional characteristics like
colour or numbthe concept of genome editing is not limited
to genes.
er of blooms in flowering plants
some disease traits in animals and plants
12. • GENOMEEDITING :This approach is called reverse genetics
• Among the key requirements of reverse genetic analysis is the ability
to modify the DNA sequence of the target organisms.
• Genome editing was selected by Nature Methods as the 2011
Method of the Year. The CRISPR-Cas system was selected by as 2015
Breakthrough of the Year
13. Pathogens weapon Can be neutralized by
Pathogen produce virulence
factor
Suppression of Virulence Functions
Plants have susceptibility gene Genome editing
Many pathogens attack crop Introduction of novel R genes and stacking
multiple genes at a preferred site.
Pathogen has defeated many R
gene
Restore defeated R genes
Ways to produce disease resistance plant
(Sebastian, 2013)
15. Comparison between traditional and modern genome editing
technologies
Mutagen Chemical(e.g., EMS) Physical (e.g., gamma,
X- ray or fast neutron
radiation)
Biological (ZFNs,
TALENs or CRISPR/ Cas)
Biological- Transgenics
(e.g., Agro or gene
gun)
Characteristic
s of genetic
variation
Substitution and Deletion Deletion and
chromosomal
mutation
Substitution and
Deletion and insertion
Insertions
Loss of function Loss of function Loss of function and gain
of function
Loss of function and gain
of function
Advantages Not necessary of knowing
gene function or sequences
Not necessary of
knowing gene function
or sequences
Gene specific mutation Insertion of genes of
known functions into
host plant genome
Easy production of random
mutation
Easy production of
random mutation
Efficient production of
desirable mutation
Efficient creation of plants
with desirable traits
9
16. Mutagen Chemical(e.g., EMS) Physical (e.g., gamma,
X- ray or fast neutron
radiation)
Biological (ZFNs,
TALENs or CRISPR/
Cas)
Biological-
Transgenics (e.g.,
Agro or gene gun)
Disadvantages Inefficient screening of
desirable traits
Inefficient screening
of desirable traits
Necessity of
knowing gene
function and
sequences
Necessity of
knowing gene
function and
sequences
Non specific mutation Non
specific
mutation
Prerequisite of
efficient
genetic
transformatio
n
Prerequisite of
efficient
genetic
transformatio
n
Other features Non transgenic
process and traits
Non transgenic
process and
traits
Transgenic
process but non
transgenic traits
Transgenic
process and
traits
10
17. GENOMEEDITING
• Genome editing, or genome editing with engineered
nucleases (GEEN) is a type of genetic engineering in which DNA is
inserted, replaced, or removed from a genome using artificially
engineered nucleases, or "molecular scissors”.
• The nucleases create specific double-strand breaks (DSBs) at desired
locations in the genome and harness the cell’s endogenous
of homologous recombination (HR)
mechanisms to repair the induced break by natural processes
and non-homologous end-
joining (NHEJ).
11
18. Non-homologous end-joining (NHEJ) Homologous recombination (HR)
Rejoins the broken ends and is often
accompanied by loss/gain of some
nucleotides
Repair DNA as a template to restore the DSBs
Thus the outcome of NHEJ is variable Outcome of this kind of repair is precise and
controllable
(Hyongbum, 2014)
19. Why genome editing?
To understand the function of a gene or a protein, one interferes with it in a
sequence-specific way and monitors its effects on the organism.
In some organisms, it is difficult or impossible to perform site-specific
mutagenesis, and therefore more indirect methods must be used, such as silencing
the gene of interest by short RNA interference (siRNA).
But sometime gene disruption by siRNA can be variable or incomplete.
Nucleases such as CRISPR can cut any targeted position in the genome and
introduce a modification of the endogenous sequences for genes that are
impossible to specifically target using conventional RNAi.
13
20. Requirement :
Ahoming device: for specific identification of target sequence
An endonuclease: for creating double strand break
Uses:
Gene knock out
Gene knock in
Gene tagging
Specific mutation (insertion/deletion study)
Promoter study
5
22. 1. Mega Nuclease
First tool used for double strand break-induced genome manipulation
Occur naturally in Yeast and in Chlamydomonas
In these enzymes binding site and restriction site occur within same unit
hence difficult to modify
Crop where it is used
Crop/plant Trait Reference
Maize Herbicide resistance Gao et al, 2010
Cotton Herbicide resistance
Insect resistance
D’Halluin et al., 2013
Limitation
-Difficult to manipulate the DNA binding site
-Small recognition site
23. 2. Zinc finger nuclease
Zinc finger protein
They were first identified as a DNA-binding motif in
Transcription factor TFIIIA from African clawed frog
(Xenopus laevis)
Small protein structural motif that is characterized by
the coordination of one or more zinc ions in order to stabilize the
fold
contain multiple finger-like protrusions that make tandem
contacts with their target molecule
These are hybrid restriction enzymes
Zn H
H
C
C
Consist of two parts: generated by fusing a zinc finger DNA-binding
domain to a DNA-cleavage domain
24. FokI, naturally found in Flavobacterium okeanokoites
N-terminal binding domain and a non-
specific DNA cleavage domain at the C-
terminal
Fok1
25. DEVELOPMENT OF ZINC FINGER NUCLEASE
Zinc finger domains can be engineered to target specific
desired DNA sequences and this enables zinc-finger
nucleases to target unique sequences within complex genomes.
By taking advantage of endogenous DNA repair machinery,
these reagents can be used to precisely alter the genomes of
higher organisms.
26. Mode of action
1.Binding of ZFN to DNA
5’
3’
2.Restricting the DNA
3’
5’
3.Cut sequence may be deleted/new
sequence may be added
+
-
4.Break end will
be sealed by host
own repairing
mechanism
27. Crop where it was used
Crop/plant Trait Rererence
Maize Herbicide tolerance Shukla et al., 2009
Soybean Physiological trait Curtin et al., 2011
Tomato against TYLCV Takenaka et al., 2007
12
Limitation
Off target effect
Construction is cumbersome and time consuming
28. 3. Transcription activator like effector nucleases (TALENs)
Bacterial cell
Plant cell
Nucleus
Consist of TALE + Endonuclease
First time reported by Ulla Bonas in Xanthomonas
oryzae (1989)
Effector
Prof. Ulla Bonas
Divert metabolic
machinery of host
towards the
pathogen
15
29. Transcription activator-like effector nucleases (TALEN) are
restriction enzymes that can be engineered to cut specific sequences of
DNA.
They are made by fusing a TAL effector DNA-binding domain to a DNA
cleavage domain (a nuclease which cuts DNA strands). Transcription
activator-like effectors (TALEs) can be engineered to bind practically any
desired DNA sequence, so when combined with a nuclease, DNA can be cut
at specific locations.
TAL (transcription activator-like) effectors are proteins secreted by
Xanthomonas bacteria via their type III secretion system when they
infect various plant species. These proteins can bind promoter sequences in
the host plant and activate the expression of plant genes that aid
bacterial infection.
30. An N-terminal domain
containing a type III
secretion signal
A central repeat domain
that determines DNA-
binding specificity
TALEs are organized into three sections
a C-terminal
containing a
domain
nuclear
localization signal and an
acidic activation domain
Astretch of 34 amino acid repeated at 15.5 - 19.5 times
…
Repeat variable diresidues (RVD)
12 13
……… ……
34 amino acid
The amino acid identity of the RVDs is responsible for DNA nucleotide recognition,
enabling the design of TALENs to target unique DNAsequences
In each repeat amino acid at the position 12 and 13 varies thus form a Repeat
variable diresidues(RVDs)
Molecular structure of effector
31. Once a DNA target is identified, an RVD for each target base is selected
according to the following code:
Designing TALEN
T
G
NG
NN, NH, NK
DNAbase
A
Amino acid in TALE
NI
C HD
Bio-informatic tools available for predicting Binding site
Programme Website Refernece
Target Finder (https://tale-nt.cac.cornell.edu/) Doyle et al., 2012
Talvez (http://bioinfo.mpl.ird.fr/cgibin/talvez/talvez.cgi) Pérez-Quintero et al., 2013
Storyteller (http://bioinfoprod.mpl.ird.fr/xantho/tales) Pérez-Quintero et al., 2013
(Mussolino and Cathomen, 2012)
32. Mode of action
Fok1
Fok1
5’ 3’
3’ 5’
+
_
1. Binding of TALEN
2. Cutting at
target site
3 In/del
5’ 3’
3’ 5’
4. Gap sealing
33. Different ways by TALENs can be use for Disease resistance plant
• Transcription activation of different R gene
• Transcription Repression of different Susceptibility Gene
• Mutation in Promoters of Susceptibility Genes
• Gene replacement
• Destroying pathogen genome
35. CRISPR–Cassystems
• These are the part of the Bacterial immune system which detects and
recognize the foreign DNA and cleaves it.
1. THE CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)
loci
2. Cas (CRISPR- associated) proteins can target and cleave invading DNA in a
sequence – specific manner.
A CRISPR array is composed of a series of repeats interspaced by spacer
sequences acquired from invading genomes.
29
36. 1987
• Researchers find CRISPR sequences in Escherichia coli, but do not
characterize their function.
2000
• CRISPR sequence are found to be common in other microbes.
2002
• Coined CRISPR name, defined signature Cas genes
2007
• First experimental evidence for CRISPR adaptive immunity
2013
• First demonstration of Cas9 genome engineering in eukaryotic cell
HISTORY
38. Different Cas proteins and their function
Protein Distribution Process Function
Cas1 Universal Spacer acquisition DNAse, not sequence specfic, can bind RNA; present in all Types
Cas2 Universal Spacer acquisition specific to U-rich regions; present in all Types
Cas3 Type I signature Target interference DNA helicase, endonuclease
Cas4 Type I, II Spacer acquisition RecB-like nuclease with exonuclease activity homologous to RecB
Cas5 Type I crRNA expression RAMP protein, endoribonuclease involved in crRNA biogenesis; part of CASCADE
Cas6 Type I, III crRNA expression RAMP protein, endoribonuclease involved in crRNA biogenesis; part of CASCADE
Cas7 Type I crRNA expression RAMP protein, endoribonuclease involved in crRNA biogenesis; part of CASCADE
Cas8 Type I crRNA expression Large protein with McrA/HNH-nuclease domain and RuvC-like nuclease; part of
CASCADE
Cas9 Type II signature Target interference Large multidomain protein with McrA-HNH nuclease domain and RuvC-like
nuclease domain; necessary for interference and target cleavage
Cas10 Type III signature crRNA expression
and interference
HD nuclease domain, palm domain, Zn ribbon; some homologies with CASCADE
elements
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39. Protospacer adjacent motif (PAM) is a DNA sequence
immediately following the DNA sequence targeted by
the Cas9 nuclease in the CRISPR bacterial adaptive immune system.
PAM is a component of the invading virus or plasmid, but is not a
component of the bacterial CRISPR locus. Cas9 will not successfully
bind to or cleave the target DNA sequence if it is not followed by the
PAM sequence.
PAM is an essential targeting component (not found in bacteria)
which distinguishes bacterial self from non-self DNA, thereby
preventing the CRISPR locus from being targeted and destroyed by
nuclease
41. Trans-activating crRNA (Tracr) RNA
A trans-encoded small RNA with 24 nucleotide complementarity to the repeat
regions of crRNAprecursor transcripts
Function
Pair with Cr RNA for its maturation by processing through RNAseIII
Activating Cr RNA-guided cleavage by cas 9
42. Action of CRISPR in bacteria
The CRISPR immune system works to protect bacteria from repeated
viral attack via three basic steps:
(1) Adaptation
(2) Production of cr RNA
(3) Targeting
36
45. On line designing tools
Software Work
ZiFit
(http://zifit.partners.org/ZiFiT/)
Helps to construct gRNAs,
TALENs, and ZFNs targeting the
sequence of interest
CRISPR designing tools
(http://crispr.mit.edu/)
Helps design gRNA sequences
that are predicted to minimize
off-target mutations
E-CRISP (http://e-crisp-
test.dkfz.de/E-CRISP/
index.html)
Permits the finding of paired
gRNAs and off targets
CRISPR-PLANT Database
(http://www.genome.arizona.edu/
An online tool that includes more
plant genomes
crispr/index.html) On line discussion group
(https://groups.google.com/forum/#!forum/talengi- neering;
(https://groups.google.com/forum/#!forum/crispr).
27
49. Examples of crops modified with CRISPR technology
43
CROPS DESCRIPTION REFERNCES
Corn Targeted mutagenesis Liang et al. 2014
Rice Targeted mutagenesis Belhaj et al. 2013
Sorghum Targeted gene modification Jiang et al. 2013b
Sweet orange Targeted genome editing Jia and Wang 2014
Tobacco Targeted mutagenesis Belhaj et al. 2013
Wheat Targeted mutagenesis Upadhyay et al. 2013, Yanpeng et
al. 2014
Potato
Soybean
Targeted mutagenesis
Gene editing
Shaohui et al., 2015
Yupeng et al., 2015
Harrison et al., 2014
50. Application in Agriculture
Can be used to create high degree of genetic variability at precise locus in the
genome of the crop plants.
Potential tool for multiplexed reverse and forward genetic study.
Precise transgene integration at specific loci.
Developing biotic and abiotic resistant traits in crop plants.
Potential tool for developing virus resistant crop varieties.
Can be used to eradicate unwanted species like herbicide resistant weeds, insect
pest.
Potential tool for improving polyploid crops like potato and wheat.
44
51. Some pitfalls of this technology
Proper selection of gRNA
Use dCas9 version of Cas9 protein
Make sure that there is no mismatch within
the seed sequences(first 12 nt adjacent to
PAM)
Use smaller gRNAof 17 nt instead of 20 nt
Sequence the organism first you want to
work with
Use NHEJ inhibitor in order to boost up
HDR 45
Solutions
Off target indels
Limited choice of PAM sequences
52. How to avoid off-target effects?
- Optimization of Injection conditions
(less cas9/sgRNA)
- Bioinformatics : Find a sgRNA target
for less off-targets “CRISPR Design”
(http://crispr.mit.edu)
46
53. Final Conclusion
Crop improvement requires the constant creation and use of new
allelic variants
Genetic modification, including plant breeding, has been widely
used to improve crop yield and quality, as well as to increase
disease resistance
Progress in site specific nuclease coupled with increase crop
genome sequencing and more effective transformation system
offers great promise in creating non transgenic crops with
predetermined trait
TALENs, CRISPR/Cas, and ZFN can be easily fashioned to bind
any specific sequence of DNA (TALEs, CRISPR/Cas) because of
the simple rules governing their interactions with nucleic acids.
54. Using these technologies ( ZFNs, TALENs, and
CRISPR/Cas9 ) plant genome can be successfully modified
Among the different genome editing tools after TALEN,
CRISPER/Cas9 is getting more popularity owing to specticity
simplicity ease in construction
A step ahead from the fear of transgenic.
Final Conclusion