Doane Chilcoat, Director, Applied Technology Systems, DuPont Pioneer
CRISPR-Cas as an advanced plant breeding tool is a more efficient way to improve plants and help farmers produce more and better food, with fewer resources. The superior properties of CRISPR-Cas allows DuPont Pioneer scientists to develop innovative and sustainable seed products for growers similar to those realized through conventional plant breeding, but with even greater efficiency, accuracy and quality. Pioneer is leading the application of this tool to develop customized agriculture solutions. In this talk, potential product targets of this promising technology will be discussed. Approaches to fostering social license and developing an open innovation model for CRISPR-Cas will also be reviewed.
Progress and prospects in plant genome editingAnilkumar C
The document summarizes a seminar on plant genome editing tools. It discusses various tools for targeted genome manipulation including zinc finger nucleases, TALENs, and CRISPR/Cas9. It provides examples of each tool being used to generate disease resistance in crops like rice and wheat. It also discusses factors like design, efficiency, and off-target effects of the different tools. Case studies demonstrate using these tools to edit susceptibility genes in rice for bacterial blight resistance and three MLO genes in wheat for powdery mildew resistance.
Allele mining in orphan underutilized cropsCCS HAU, HISAR
This document discusses allele mining as a research field aimed at identifying allelic variation in genetic resources collections that can be used for crop improvement. It defines key terms like alleles, orphan crops, and describes two major approaches for allele mining - TILLING and sequencing-based methods. Case studies on allele mining in cassava and sorghum are presented, outlining methodology used and results obtained, including the identification of superior alleles. The prospects of allele mining in molecular plant breeding are discussed, and the need for standardizing bioinformatics tools and developing advanced strategies to efficiently identify novel alleles from genetic resources.
Genome editing technologies allow genetic material to be added, removed or altered at specific locations in an organism's genome. Several approaches exist, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR/Cas9, and base editors. These tools create precise breaks in DNA that can be repaired through non-homologous end joining or homology-directed repair. They enable trait discovery and crop improvement by generating plants with high yield, stress resistance, or other desired properties. While powerful, challenges remain in fully editing complex genomes and reducing off-target mutations.
Marker Assisted Gene Pyramiding for Disease Resistance in RiceIndrapratap1
Why marker assisted gene pyramiding?
For traits that are simply inherited, but that are difficult or expensive to measure phenotypically, and/or that do not have a consistent phenotypic expression under specific selection conditions, marker-based selection is more effective than phenotypic selection.
Traits which are traditionally regarded as quantitative and not targeted by gene pyramiding program can be improved using gene pyramiding if major genes affecting the traits are identified.
Genes with very similar phenotypic effects, which are impossible or difficult to combine in single genotype using phenotypic selection, can be pyramided through marker assisted selection.
Markers provides a more effective option to control linkage drag and make the use of genes contained in unadapted resources easier.
Pyramiding is possible through conventional breeding but is extremely difficult or impossible at early generations..
DNA markers may facilitate selection because DNA marker assays are non destructive and markers for multiple specific genes/QTLs can be tested using a single DNA sample without phenotyping.
CONCLUSION:
• Molecular marker offer great scope for improving the efficiency of conventional plant breeding.
• Gene pyramiding may not be the most suitable strategy when many QTL with small effects control the trait and other methods such as marker-assisted recurrent selection should be considered.
• With MAS based gene pyramiding, it is now possible for breeder to conduct many rounds of selections in a year.
• Gene pyramiding with marker technology can integrate into existing plant breeding program all over the world to allow researchers to access, transfer and combine genes at a rate and with precision not previously possible.
• This will help breeders get around problems related to larger breeding populations, replications in diverse environments, and speed up the development of advance lines.
For further queries please contact at isag2010@gmail.com
This document discusses genetic and molecular markers. It defines genetic markers as any phenotypic difference controlled by genes that can be used to study inheritance or selection of linked genes. Molecular markers are readily detectable DNA sequences whose inheritance can be monitored. The document describes different types of molecular markers including protein markers, RFLP, RAPD, AFLP, microsatellites, SNPs, VNTRs and STRs. It provides details on how each marker type is detected and analyzed, their advantages and disadvantages, and applications in genetics research.
This document discusses genome editing in fruit crops using CRISPR/Cas9 technology. It provides examples of using CRISPR to edit genes involved in fruit ripening, pigmentation, and flowering time regulation in strawberry, banana, apple, and kiwifruit. Specifically, it describes using CRISPR to increase beta-carotene levels in banana, induce early flowering in apple and pear, and generate dwarf kiwifruit plants. The document concludes that integrating biotechnology like CRISPR with conventional breeding is a promising strategy for fruit crop improvement.
Progress and prospects in plant genome editingAnilkumar C
The document summarizes a seminar on plant genome editing tools. It discusses various tools for targeted genome manipulation including zinc finger nucleases, TALENs, and CRISPR/Cas9. It provides examples of each tool being used to generate disease resistance in crops like rice and wheat. It also discusses factors like design, efficiency, and off-target effects of the different tools. Case studies demonstrate using these tools to edit susceptibility genes in rice for bacterial blight resistance and three MLO genes in wheat for powdery mildew resistance.
Allele mining in orphan underutilized cropsCCS HAU, HISAR
This document discusses allele mining as a research field aimed at identifying allelic variation in genetic resources collections that can be used for crop improvement. It defines key terms like alleles, orphan crops, and describes two major approaches for allele mining - TILLING and sequencing-based methods. Case studies on allele mining in cassava and sorghum are presented, outlining methodology used and results obtained, including the identification of superior alleles. The prospects of allele mining in molecular plant breeding are discussed, and the need for standardizing bioinformatics tools and developing advanced strategies to efficiently identify novel alleles from genetic resources.
Genome editing technologies allow genetic material to be added, removed or altered at specific locations in an organism's genome. Several approaches exist, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR/Cas9, and base editors. These tools create precise breaks in DNA that can be repaired through non-homologous end joining or homology-directed repair. They enable trait discovery and crop improvement by generating plants with high yield, stress resistance, or other desired properties. While powerful, challenges remain in fully editing complex genomes and reducing off-target mutations.
Marker Assisted Gene Pyramiding for Disease Resistance in RiceIndrapratap1
Why marker assisted gene pyramiding?
For traits that are simply inherited, but that are difficult or expensive to measure phenotypically, and/or that do not have a consistent phenotypic expression under specific selection conditions, marker-based selection is more effective than phenotypic selection.
Traits which are traditionally regarded as quantitative and not targeted by gene pyramiding program can be improved using gene pyramiding if major genes affecting the traits are identified.
Genes with very similar phenotypic effects, which are impossible or difficult to combine in single genotype using phenotypic selection, can be pyramided through marker assisted selection.
Markers provides a more effective option to control linkage drag and make the use of genes contained in unadapted resources easier.
Pyramiding is possible through conventional breeding but is extremely difficult or impossible at early generations..
DNA markers may facilitate selection because DNA marker assays are non destructive and markers for multiple specific genes/QTLs can be tested using a single DNA sample without phenotyping.
CONCLUSION:
• Molecular marker offer great scope for improving the efficiency of conventional plant breeding.
• Gene pyramiding may not be the most suitable strategy when many QTL with small effects control the trait and other methods such as marker-assisted recurrent selection should be considered.
• With MAS based gene pyramiding, it is now possible for breeder to conduct many rounds of selections in a year.
• Gene pyramiding with marker technology can integrate into existing plant breeding program all over the world to allow researchers to access, transfer and combine genes at a rate and with precision not previously possible.
• This will help breeders get around problems related to larger breeding populations, replications in diverse environments, and speed up the development of advance lines.
For further queries please contact at isag2010@gmail.com
This document discusses genetic and molecular markers. It defines genetic markers as any phenotypic difference controlled by genes that can be used to study inheritance or selection of linked genes. Molecular markers are readily detectable DNA sequences whose inheritance can be monitored. The document describes different types of molecular markers including protein markers, RFLP, RAPD, AFLP, microsatellites, SNPs, VNTRs and STRs. It provides details on how each marker type is detected and analyzed, their advantages and disadvantages, and applications in genetics research.
This document discusses genome editing in fruit crops using CRISPR/Cas9 technology. It provides examples of using CRISPR to edit genes involved in fruit ripening, pigmentation, and flowering time regulation in strawberry, banana, apple, and kiwifruit. Specifically, it describes using CRISPR to increase beta-carotene levels in banana, induce early flowering in apple and pear, and generate dwarf kiwifruit plants. The document concludes that integrating biotechnology like CRISPR with conventional breeding is a promising strategy for fruit crop improvement.
This document summarizes information from a student's assignment on plant genome sequencing techniques. It discusses early phenotypic selection methods and their limitations. It then summarizes different sequencing strategies used for important crop plants like rice, poplar, and Arabidopsis. These include BAC-by-BAC, whole genome shotgun, and various next-generation sequencing platforms. The document also summarizes applications of sequencing including identifying genes related to rice yield and flowering time and using sequencing to improve potato and maize varieties.
Molecular markers and Functional molecular markersChandana B.R.
This document discusses functional markers and their development and use in plant breeding. It begins by defining markers and describing different types of markers used historically, from morphological to molecular markers. It then focuses on functional markers, which are derived from polymorphisms within genes that affect traits of interest. The document discusses different types of functional markers like SSR and SNP-based markers. It notes advantages of functional markers include not requiring validation and providing direct information about gene effects. Limitations include that many genes have not been functionally characterized. The document ends with a case study using EST-SSR markers to estimate genetic diversity in maize breeding populations.
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.
RNAi – Mechanism and Its Application In Crop Improvementkundan Jadhao
This document summarizes an RNAi presentation on crop improvement using RNA interference. The 3-sentence summary is:
RNA interference (RNAi) is a process of post-transcriptional gene silencing mediated by small RNA molecules. The presentation described the RNAi pathway and various applications of RNAi technology in crop improvement, including increasing nutrient levels, developing virus and pest resistance, and reducing anti-nutritional compounds. Several case studies were provided that demonstrated how RNAi has been used to successfully modify traits in different crops like maize, cotton, coffee, and banana.
Single Nucleotide Polymorphism Genotyping Using Kompetitive Allele Specific ...MANGLAM ARYA
Single Nucleotide Polymorphism
Single nucleotide polymorphism (SNP) refers to a single base change in a DNA sequence
SNP: Commonly biallelic
Two types(Based on presence in genome)
Synonymus
Non-synonymus
SNPs have largely replaced simple sequence repeats (SSRs)
Advantage of using SNPs
Low assay cost
High genomic abundance
Locus specificity
co-dominant inheritance
Simple documentation
Potential for high-throughput Analysis
Relatively low genotyping error rates
SNP genotyping platforms
BeadXpressTM,GoldenGateTM and Infinium from Illumina
GeneChipTM and GenFlexTM Tag array from Affimetrix
SNaPshotTM and TaqManTM from the Applied Biosystems
SNPWaveTM from KeyGene
iPLEX GoldTM Assay and Mass-RRAYTM from Sequonome
Variables to be considered
Throughput
Data turnaround
Time
Ease of use
Performance (sensitivity, reliability, reproducibility, and accuracy),
Flexibility (genotyping few samples with many snps or many samples with few snps),
Number of markers generated per run (uniplex versus multiplex assay capability)
Assay development requirements and genotyping cost per sample or data point.
KASP
KBioscience Competitive Allele-Specific PCR
Homogenous, Fluorescence-based genotyping technology, based on
Allele-specific oligo extension (primer)
Fluorescence resonance energy transfer
KASP Applications
Genotyping a wide range of species for various purposes.
KASP for Quality analysis, QTL mapping, MARS, and allele mining
Quality Control Analysis
QC analysis should be done for two reasons by genotyping the parents and F1s with the same subset of SNPs, in order to
confirm if F1s contains true-to-type alleles from their parents
check the genetic purity of the inbred parents.
F1s with true-to-type parental alleles for at least 90 % of the SNPs that were polymorphic between the parents should be advanced, while those with less than 10 % nonparental alleles should be discarded.
QTL Mapping
QTL mapping identifies a subset of markers that are significantly associated with one or more QTL influencing the expression of the trait of interest.
1) Select or develop a bi-parental mapping population.
2) Phenotype the population for a trait under greenhouse or field conditions.
3) Choose a molecular marking system – genotype parents of the mapping population and F1s with large numbers of markers, then select 200-400 markers exhibiting polymorphism between the parents.
4) Choose a genotyping approach, then generate molecular data for polymorphic markers
5) Identify the molecular markers associated with major QTL using statistical programs.
Large-scale allele mining
Allele mining is a promising approach to dissecting naturally occurring allelic variation at candidate genes controlling key agronomic traits.
KASP platform at CIMMYT has been used for the systematic mining of large germplasm collections for specific functional polymorphisms.
SNPs or small indels that
Gene silencing, also known as RNA interference, is a natural process in plants that evolved as a defense mechanism against viruses. Transgene silencing occurs when introduced transgenes are not expressed due to this silencing process. The first evidence of this was discovered in 1990 by R. Jorgensen in petunia plants, where both an introduced gene and endogenous gene were silenced. Gene silencing can occur at the transcriptional or post-transcriptional level through mechanisms like siRNA and microRNA production. Virus-induced gene silencing is a technique used to study gene function and develop virus-resistant plants by suppressing viral gene expression. Applications of gene silencing include developing disease-resistant crops and modifying plant traits.
i explained about basics of genome engineering and crispr system.
CRISPR will change the world and it is just the beginning, are you ready to meet the future? you think its great and beautiful or.....?
please give your feedback to my email
pooyanaghshbandi@yahoo.com
i am starting to write a critical and fantastic review article about CRISPR, if you are interested to join please contact me.
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.
Multiple inbred founder lines are inter-mated for several generations prior to creating inbred lines, resulting in a diverse population whose genomes are fine scale mosaics of contributions from all founders.
Research Program Genetic Gains (RPGG) Review Meeting 2021: Forward Breeding: ...ICRISAT
Integration of various molecular breeding approaches (MABC, MARS, and GS) in the product development process at ICRISAT. Accelerated rate of genetic gain across all mandate crops by leveraging expertise from various groups inside and outside ICRISAT.
Development of climate resilient crops / agricultureAmol Gaikwad
This document discusses developing climate-resilient crops through harnessing genetic variability. It begins with an introduction to climate change impacts on agriculture and the importance of breeding resilient crops. It then explores genetic variability as the foundation for crop improvement, describing sources of variability like natural mutations, crossbreeding, and genetic engineering. Common breeding approaches are discussed, such as mutation breeding, hybridization, marker-assisted selection, and genetic engineering. Examples are given of climate-resilient crops developed through these methods, including drought-tolerant maize, heat-tolerant wheat, flood-tolerant rice, and salinity-tolerant crops. The document concludes by emphasizing how genetic variability enables developing crops adapted to changing climates,
Molecular Marker-assisted Breeding in RiceFOODCROPS
1. The document discusses molecular marker-assisted breeding in rice. It provides details on the expertise and experiences of Dr. Jian-Long Xu in molecular rice breeding including allele mining and marker-assisted selection.
2. Marker-assisted selection is described as a method to select phenotypes based on the genotype of linked markers rather than the target gene itself. The advantages of MAS include time and cost savings compared to traditional field trials.
3. Requirements for large-scale application of MAS include validation of QTL in breeding materials, efficient genotyping protocols, and decision support tools for breeders.
Reverse Breeding: a tool to create homozygous plants from the heterozygous po...Sanjay Kumar
This document provides an overview of reverse breeding, a novel plant breeding technique to directly produce homozygous parental lines from any heterozygous plant. It discusses how reverse breeding uses RNA interference to suppress meiotic recombination and produce doubled haploids from gametes, generating homozygous parental lines. The document summarizes a case study applying this to Arabidopsis thaliana and discusses applications like reconstructing hybrids, breeding at the chromosome level, and implications for food safety. Limitations and future research directions are also outlined.
Marker assisted breeding of biotic stress resistance in Rice Senthil Natesan
A marker is a DNA sequence which serves as a signpost/flag post
linked to the trait/gene of interest and is co-inherited along with
the trait
Presence of specific allele of marker = Presence of specific allele of target gene based on the concept the MAS practiced -R.M. Sundaram
Directorate Rice of Research, Hydrabad , July 3rd 2009, CPMB&B, TNAU presentation
CRISPR/Cas9 is an advanced genome editing technology that can be used to develop plant disease resistance. It involves a Cas9 enzyme that acts like molecular scissors to cut DNA at specific locations guided by CRISPR RNA. This triggers DNA repair that can introduce changes to genes. Researchers have used CRISPR/Cas9 to develop resistance in plants against viruses, fungi, and bacteria by editing genes involved in host-pathogen interaction and disease susceptibility. It provides a precise and efficient way to edit plant genomes to improve crop resistance compared to previous tools. Scientists continue working to enhance the specificity and control of CRISPR/Cas9 for genome editing applications in agriculture.
CRISPR-Cas is a natural defense system in bacteria that uses CRISPR sequences and Cas proteins to target and degrade foreign DNA such as from viruses. It has been adapted for genome editing in other organisms using a Cas9 protein guided by a synthetic single guide RNA to introduce targeted double-strand breaks. This system allows for precise genome modifications and has applications in biomedical research, disease treatment, and engineering of plants and other organisms. However, off-target effects and delivery methods require further optimization.
Microsatellite are powerful DNA markers for quantifying genetic variations within & between populations of a species, also called as STR, SSR, VNTR. Tandemly repeated DNA sequences with the repeat/size of 1 – 6 bases repeated several times
Potential for off-target effects in topically applied dsRNA-based products us...OECD Environment
10-12 April 2019: The OECD Conference on RNAi based pesticides provided an overview on the current status and future possibilities for the regulation of externally applied dsRNA-based products that are proposed for use as pesticides. The event facilitated exchanges between policy makers, academia, industry on their implications in health, environment, and regulation.
This document summarizes Mohd Kyum's PhD research on using CRISPR/Cas9 genome editing to induce haploidy in rice. The objectives were to generate knockout mutants of the OsMATL gene, which is responsible for haploid induction in maize, using CRISPR/Cas9 RNP complexes delivered via particle bombardment. Embryos of the rice variety Purple Line were bombarded with three different sgRNAs targeting OsMATL. Regenerated T0 plants were analyzed for mutations in the target gene using techniques like T7E1 assay, PCR-RFLP, and sequencing. Putative mutant plants showed a transformation efficiency of around 12.5% and are being further analyzed to characterize mutations induced
Recent breakthroughs in genome editing technology have led to a rapid adoption that parallels that seen with RNAi. And like RNAi, these methods are taking the scientific world by storm, with high profile publications in fields as diverse as HIV treatment, stem cell therapy, food crop modification and drug development to name but a few.
Critically, the endogenous modification of genes enables the study of their function in a physiological context. It also overcomes some of the artefacts that can result from established techniques such as transgenesis and RNAi, which have mislead researchers with false positives or negatives. Until recently however genome editing required considerable technical expertise, and consequently was a relatively niche pursuit.
In this talk we will look at how the latest developments in genome editing tools have changed this, with improvements in both ease-of-use and targeting efficiency, as well as a concomitant reduction in costs opening up these approaches to the wider scientific community.
Rapid adoption of the CRISPR/Cas9 system has for example led to a long list of organisms and tissues in which genetic changes have been made with high efficiency. Other technologies such as recombinant adeno-associated virus (rAAV) offer further precision, stimulating the cell’s high-fidelity DNA repair pathways to insert exogenous sequence with unrivalled specificity. Targeting efficiency can be improved still further by using the technologies in combination – genome cutting induced by CRISPR can significantly enhance homologous recombination mediated by rAAV.
Despite these rapid advances, some pitfalls remain, and so we’ll discuss some of the key considerations for avoiding these, ranging from simply picking the right tool for the job to designing an experiment that maximises chances of success.
Finally we’ll look at how genome editing is being applied to both basic and translational research, and in both a gene-specific and genome wide manner. For the study of disease associated genes and mutations scientists can now complement wide panels of tumour cells with genetically defined isogenic cell pairs identical in all but precise modifications in their gene of interest. The ease-of-design and efficiency of the CRISPR system is also being exploited for genome wide synthetic lethality screens, facilitating rapid drug target identification with significantly reduced risk of false negatives and off-target false positives. And again, further synergies are achieved when these approaches are combined to look for potential synthetic lethal targets in specific genomic contexts.
This document summarizes information from a student's assignment on plant genome sequencing techniques. It discusses early phenotypic selection methods and their limitations. It then summarizes different sequencing strategies used for important crop plants like rice, poplar, and Arabidopsis. These include BAC-by-BAC, whole genome shotgun, and various next-generation sequencing platforms. The document also summarizes applications of sequencing including identifying genes related to rice yield and flowering time and using sequencing to improve potato and maize varieties.
Molecular markers and Functional molecular markersChandana B.R.
This document discusses functional markers and their development and use in plant breeding. It begins by defining markers and describing different types of markers used historically, from morphological to molecular markers. It then focuses on functional markers, which are derived from polymorphisms within genes that affect traits of interest. The document discusses different types of functional markers like SSR and SNP-based markers. It notes advantages of functional markers include not requiring validation and providing direct information about gene effects. Limitations include that many genes have not been functionally characterized. The document ends with a case study using EST-SSR markers to estimate genetic diversity in maize breeding populations.
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.
RNAi – Mechanism and Its Application In Crop Improvementkundan Jadhao
This document summarizes an RNAi presentation on crop improvement using RNA interference. The 3-sentence summary is:
RNA interference (RNAi) is a process of post-transcriptional gene silencing mediated by small RNA molecules. The presentation described the RNAi pathway and various applications of RNAi technology in crop improvement, including increasing nutrient levels, developing virus and pest resistance, and reducing anti-nutritional compounds. Several case studies were provided that demonstrated how RNAi has been used to successfully modify traits in different crops like maize, cotton, coffee, and banana.
Single Nucleotide Polymorphism Genotyping Using Kompetitive Allele Specific ...MANGLAM ARYA
Single Nucleotide Polymorphism
Single nucleotide polymorphism (SNP) refers to a single base change in a DNA sequence
SNP: Commonly biallelic
Two types(Based on presence in genome)
Synonymus
Non-synonymus
SNPs have largely replaced simple sequence repeats (SSRs)
Advantage of using SNPs
Low assay cost
High genomic abundance
Locus specificity
co-dominant inheritance
Simple documentation
Potential for high-throughput Analysis
Relatively low genotyping error rates
SNP genotyping platforms
BeadXpressTM,GoldenGateTM and Infinium from Illumina
GeneChipTM and GenFlexTM Tag array from Affimetrix
SNaPshotTM and TaqManTM from the Applied Biosystems
SNPWaveTM from KeyGene
iPLEX GoldTM Assay and Mass-RRAYTM from Sequonome
Variables to be considered
Throughput
Data turnaround
Time
Ease of use
Performance (sensitivity, reliability, reproducibility, and accuracy),
Flexibility (genotyping few samples with many snps or many samples with few snps),
Number of markers generated per run (uniplex versus multiplex assay capability)
Assay development requirements and genotyping cost per sample or data point.
KASP
KBioscience Competitive Allele-Specific PCR
Homogenous, Fluorescence-based genotyping technology, based on
Allele-specific oligo extension (primer)
Fluorescence resonance energy transfer
KASP Applications
Genotyping a wide range of species for various purposes.
KASP for Quality analysis, QTL mapping, MARS, and allele mining
Quality Control Analysis
QC analysis should be done for two reasons by genotyping the parents and F1s with the same subset of SNPs, in order to
confirm if F1s contains true-to-type alleles from their parents
check the genetic purity of the inbred parents.
F1s with true-to-type parental alleles for at least 90 % of the SNPs that were polymorphic between the parents should be advanced, while those with less than 10 % nonparental alleles should be discarded.
QTL Mapping
QTL mapping identifies a subset of markers that are significantly associated with one or more QTL influencing the expression of the trait of interest.
1) Select or develop a bi-parental mapping population.
2) Phenotype the population for a trait under greenhouse or field conditions.
3) Choose a molecular marking system – genotype parents of the mapping population and F1s with large numbers of markers, then select 200-400 markers exhibiting polymorphism between the parents.
4) Choose a genotyping approach, then generate molecular data for polymorphic markers
5) Identify the molecular markers associated with major QTL using statistical programs.
Large-scale allele mining
Allele mining is a promising approach to dissecting naturally occurring allelic variation at candidate genes controlling key agronomic traits.
KASP platform at CIMMYT has been used for the systematic mining of large germplasm collections for specific functional polymorphisms.
SNPs or small indels that
Gene silencing, also known as RNA interference, is a natural process in plants that evolved as a defense mechanism against viruses. Transgene silencing occurs when introduced transgenes are not expressed due to this silencing process. The first evidence of this was discovered in 1990 by R. Jorgensen in petunia plants, where both an introduced gene and endogenous gene were silenced. Gene silencing can occur at the transcriptional or post-transcriptional level through mechanisms like siRNA and microRNA production. Virus-induced gene silencing is a technique used to study gene function and develop virus-resistant plants by suppressing viral gene expression. Applications of gene silencing include developing disease-resistant crops and modifying plant traits.
i explained about basics of genome engineering and crispr system.
CRISPR will change the world and it is just the beginning, are you ready to meet the future? you think its great and beautiful or.....?
please give your feedback to my email
pooyanaghshbandi@yahoo.com
i am starting to write a critical and fantastic review article about CRISPR, if you are interested to join please contact me.
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.
Multiple inbred founder lines are inter-mated for several generations prior to creating inbred lines, resulting in a diverse population whose genomes are fine scale mosaics of contributions from all founders.
Research Program Genetic Gains (RPGG) Review Meeting 2021: Forward Breeding: ...ICRISAT
Integration of various molecular breeding approaches (MABC, MARS, and GS) in the product development process at ICRISAT. Accelerated rate of genetic gain across all mandate crops by leveraging expertise from various groups inside and outside ICRISAT.
Development of climate resilient crops / agricultureAmol Gaikwad
This document discusses developing climate-resilient crops through harnessing genetic variability. It begins with an introduction to climate change impacts on agriculture and the importance of breeding resilient crops. It then explores genetic variability as the foundation for crop improvement, describing sources of variability like natural mutations, crossbreeding, and genetic engineering. Common breeding approaches are discussed, such as mutation breeding, hybridization, marker-assisted selection, and genetic engineering. Examples are given of climate-resilient crops developed through these methods, including drought-tolerant maize, heat-tolerant wheat, flood-tolerant rice, and salinity-tolerant crops. The document concludes by emphasizing how genetic variability enables developing crops adapted to changing climates,
Molecular Marker-assisted Breeding in RiceFOODCROPS
1. The document discusses molecular marker-assisted breeding in rice. It provides details on the expertise and experiences of Dr. Jian-Long Xu in molecular rice breeding including allele mining and marker-assisted selection.
2. Marker-assisted selection is described as a method to select phenotypes based on the genotype of linked markers rather than the target gene itself. The advantages of MAS include time and cost savings compared to traditional field trials.
3. Requirements for large-scale application of MAS include validation of QTL in breeding materials, efficient genotyping protocols, and decision support tools for breeders.
Reverse Breeding: a tool to create homozygous plants from the heterozygous po...Sanjay Kumar
This document provides an overview of reverse breeding, a novel plant breeding technique to directly produce homozygous parental lines from any heterozygous plant. It discusses how reverse breeding uses RNA interference to suppress meiotic recombination and produce doubled haploids from gametes, generating homozygous parental lines. The document summarizes a case study applying this to Arabidopsis thaliana and discusses applications like reconstructing hybrids, breeding at the chromosome level, and implications for food safety. Limitations and future research directions are also outlined.
Marker assisted breeding of biotic stress resistance in Rice Senthil Natesan
A marker is a DNA sequence which serves as a signpost/flag post
linked to the trait/gene of interest and is co-inherited along with
the trait
Presence of specific allele of marker = Presence of specific allele of target gene based on the concept the MAS practiced -R.M. Sundaram
Directorate Rice of Research, Hydrabad , July 3rd 2009, CPMB&B, TNAU presentation
CRISPR/Cas9 is an advanced genome editing technology that can be used to develop plant disease resistance. It involves a Cas9 enzyme that acts like molecular scissors to cut DNA at specific locations guided by CRISPR RNA. This triggers DNA repair that can introduce changes to genes. Researchers have used CRISPR/Cas9 to develop resistance in plants against viruses, fungi, and bacteria by editing genes involved in host-pathogen interaction and disease susceptibility. It provides a precise and efficient way to edit plant genomes to improve crop resistance compared to previous tools. Scientists continue working to enhance the specificity and control of CRISPR/Cas9 for genome editing applications in agriculture.
CRISPR-Cas is a natural defense system in bacteria that uses CRISPR sequences and Cas proteins to target and degrade foreign DNA such as from viruses. It has been adapted for genome editing in other organisms using a Cas9 protein guided by a synthetic single guide RNA to introduce targeted double-strand breaks. This system allows for precise genome modifications and has applications in biomedical research, disease treatment, and engineering of plants and other organisms. However, off-target effects and delivery methods require further optimization.
Microsatellite are powerful DNA markers for quantifying genetic variations within & between populations of a species, also called as STR, SSR, VNTR. Tandemly repeated DNA sequences with the repeat/size of 1 – 6 bases repeated several times
Potential for off-target effects in topically applied dsRNA-based products us...OECD Environment
10-12 April 2019: The OECD Conference on RNAi based pesticides provided an overview on the current status and future possibilities for the regulation of externally applied dsRNA-based products that are proposed for use as pesticides. The event facilitated exchanges between policy makers, academia, industry on their implications in health, environment, and regulation.
This document summarizes Mohd Kyum's PhD research on using CRISPR/Cas9 genome editing to induce haploidy in rice. The objectives were to generate knockout mutants of the OsMATL gene, which is responsible for haploid induction in maize, using CRISPR/Cas9 RNP complexes delivered via particle bombardment. Embryos of the rice variety Purple Line were bombarded with three different sgRNAs targeting OsMATL. Regenerated T0 plants were analyzed for mutations in the target gene using techniques like T7E1 assay, PCR-RFLP, and sequencing. Putative mutant plants showed a transformation efficiency of around 12.5% and are being further analyzed to characterize mutations induced
Recent breakthroughs in genome editing technology have led to a rapid adoption that parallels that seen with RNAi. And like RNAi, these methods are taking the scientific world by storm, with high profile publications in fields as diverse as HIV treatment, stem cell therapy, food crop modification and drug development to name but a few.
Critically, the endogenous modification of genes enables the study of their function in a physiological context. It also overcomes some of the artefacts that can result from established techniques such as transgenesis and RNAi, which have mislead researchers with false positives or negatives. Until recently however genome editing required considerable technical expertise, and consequently was a relatively niche pursuit.
In this talk we will look at how the latest developments in genome editing tools have changed this, with improvements in both ease-of-use and targeting efficiency, as well as a concomitant reduction in costs opening up these approaches to the wider scientific community.
Rapid adoption of the CRISPR/Cas9 system has for example led to a long list of organisms and tissues in which genetic changes have been made with high efficiency. Other technologies such as recombinant adeno-associated virus (rAAV) offer further precision, stimulating the cell’s high-fidelity DNA repair pathways to insert exogenous sequence with unrivalled specificity. Targeting efficiency can be improved still further by using the technologies in combination – genome cutting induced by CRISPR can significantly enhance homologous recombination mediated by rAAV.
Despite these rapid advances, some pitfalls remain, and so we’ll discuss some of the key considerations for avoiding these, ranging from simply picking the right tool for the job to designing an experiment that maximises chances of success.
Finally we’ll look at how genome editing is being applied to both basic and translational research, and in both a gene-specific and genome wide manner. For the study of disease associated genes and mutations scientists can now complement wide panels of tumour cells with genetically defined isogenic cell pairs identical in all but precise modifications in their gene of interest. The ease-of-design and efficiency of the CRISPR system is also being exploited for genome wide synthetic lethality screens, facilitating rapid drug target identification with significantly reduced risk of false negatives and off-target false positives. And again, further synergies are achieved when these approaches are combined to look for potential synthetic lethal targets in specific genomic contexts.
Genome Editing Comes of Age; CRISPR, rAAV and the new landscape of molecular ...Candy Smellie
Information is no longer a bottleneck, emphasis is shifting to the ‘what does it all mean’
In a translational context we hope that by answering that question we will be able to is to characterise the genetics that drive disease, and indeed develop drugs and diagnostics that are personalised to patients.
Genome editing provides the link between the information here, and this outcome here, by allowing scientists to recapitulate specific genetic alterations in any gene in any living tissue to probe function, develop disease models and identify therapeutic strategies. So, not only do we now have unparalleled access to genetic information, but we now have the tools to most accuartely understand what this genetic information – with genome editing allowing us to explore the genetic drivers of disease in physiological models.
AAV is a single-stranded, linear DNA virus with a a 4.7 kb genome which for the purpose of genome editing is replaced almost in entirety with the targeting vector sequence (except for the iTRs)
It is in effect a highly effective DNA delivery mechanism
After entry of the vector into the cell, target-specific homologous DNA is believed to activate and recruit HR-dependent repair factors can induce HR at rates approximately 1,000 times greater than plasmid based double stranded DNA vectors, but the mechanism by which it achieves this is still largely unknown
By including a selection cassette can select for cells that have integrated the targeting vector, and then screen for clones which have undergone targeted insetion rather than random integration, which will generally be around 1%.
Introduction and key considerations around gene-editing using CRISPR and rAAV.
With an overview of our knock-out library using the haploid cell line HAP1
Nonsyndromic orofacial clefts (NSOFCs) are the most common craniofacial malformations observed
across the globe. They are classified into three types: (a) cleft palate, (b) cleft lip, and (c) cleft lip and
palate.
Foreign genes can be expressed in both prokaryotic and eukaryotic systems. Recombinant proteins have many pharmaceutical and industrial uses. Prokaryotic expression is faster and cheaper but some eukaryotic proteins may lack proper processing. Eukaryotic systems like yeast, insect cells and mammalian cells allow for proper post-translational modifications but are more complex and expensive. The choice of expression system depends on the desired protein properties and costs involved.
Crop plants: DNA-free genome editing with CRISPR enzymesOECD Environment
This document summarizes a presentation on DNA-free genome editing with CRISPR enzymes. It discusses the evolution of genome editing techniques from random mutagenesis to targeted methods using ZFN, TALENs, and CRISPR/Cas9. The presentation focuses on a DNA-free approach using purified Cas9 protein and sgRNA to assemble CRISPR ribonucleoproteins (RNPs) for direct delivery into plant cells. It shows this method can induce targeted mutations in Arabidopsis, rice, and lettuce without leaving foreign DNA. RNPs generated mutations with high efficiency and specificity, and mutations were heritable after plant regeneration. Compared to other methods, DNA-free RNP delivery avoids issues like trans
Peter Morrell Ag Experimental Station Talk 2018PeterMorrell4
Peter L. Morrell presented the objectives and progress of his research group. The group has 7 papers assessing the impact of deleterious variants in crops, 3 papers using environmental association to identify variants for traits like cold tolerance in barley, and 2 papers on admixture approaches to study crop domestication. They also have 4 papers on linkage disequilibrium in crops and provide computational training. The group identified candidate variants for cold tolerance and found they were in regions of high linkage disequilibrium.
Making genome edits in mammalian cellsChris Thorne
Looking at the kind of modifications that can be made in mammalian cells, and how at Horizon moving to a haploid model system has significantly improved efficiency of both editing and validation
CRISPR Is On The Move: Genome Editing From Rice To WheatFabio Caligaris
Presented at Plant Genomics and Gene Editing Congress: Europe. For more information visit: www.global-engage.com
Success in rice is not equalled in wheat: CRISPR/Cas9 efficiency is around 5% and requires a time-consuming downstream identification of the genome edited plants.
Genetic engineering involves manipulating DNA through techniques like selective breeding, hybridization, genetic bottlenecks, inbreeding, and genetic engineering. Genetic engineering uses vectors to insert genes into host organisms. Key steps include isolating the gene, inserting it into a host using a vector, producing copies of the host, and purifying the gene product. Restriction enzymes and ligases are important tools that cut and join DNA. PCR is used to amplify DNA, and sequencing methods like Sanger sequencing determine the DNA sequence. Primer design is important for techniques like PCR, cloning, and discovery of unknown sequences through degenerate primers.
Lessons learned from high throughput CRISPR targeting in human cell linesChris Thorne
In just a short period of time CRISPR-Cas9 technology has revolutionized the field of genome editing, and taken the scientific community by storm. Already our understanding of how best to apply this technology has advanced significantly and almost every week new publications appear showcasing its application in basic and translational research.
While CRISPR-Cas9 is applicable across many different cell types, we have found it particularly suited for genome editing in near-haploid human cell lines. This has allowed us to establish a robust pipeline for the inactivation of non-essential genes at unprecedented scale and efficiency.
We have now knocked out over 1500 human genes and have generated a resource that is, to the best of our knowledge, the largest collection of human knockout cell lines available, covering comprehensive subsets of genes clustered by biological pathway (e.g. the autophagy pathway, the JAK/STAT pathway) or by phylogenetic relationship (e.g. kinases, bromodomain-containing proteins).
In this talk we will discuss how, through more than 1500 genome editing experiments, we have started to unravel some of the general principles governing the use of CRISPR-Cas9 in mammalian cells. For example, we have analyzed the impact of variation in the guide RNA sequence on Cas9 cleavage efficiency and characterized the mutational signature arising from CRISPR-Cas9 cleavage.
We will also highlight (with examples) how these learnings are now being applied to introduce other genomic modifications in a high throughput manner, including chromosomal deletions, translocations, point mutations and endogenous gene tags.
Doubled Haploid Production Techniques of Horticultural CropsMahbubul Hassan
This document discusses techniques for doubled haploid production in horticultural crops including cucumber and tomato. It provides details on:
1. Pollen irradiation protocols for cucumber, where pollen doses of 50-200 Gy can be used for mutagenesis and doses of 300-400 Gy kill pollen for haploid embryo development.
2. Anther culture techniques for tomato using gamma irradiation, where doses of 75-150 Gy produced the highest percentage of responsive anthers forming callus.
3. Effects of pollen irradiation on fruit set in tomato, where doses of 50-200 Gy resulted in comparable fruit set to controls, while 300-400 Gy doses did not produce any fruit set.
HIV Vaccines Process Development & Manufacturing - Pitfalls & PossibilitiesKBI Biopharma
Originally presented at the HIV Vaccine Manufacturing Workshop –July 19th& 20th, 2017 by Abhinav A. Shukla, Ph.D.Senior Vice PresidentDevelopment & ManufacturingKBI Biopharma, Durham NC
CRISPR-Cas9 : A novel tool for genome editing for crop improvementGarima188199
This document summarizes a study that used CRISPR/Cas9 genome editing to modify the SlPG gene in tomatoes, which codes for the polygalacturonase enzyme involved in fruit softening. Five sgRNAs targeting the SlPG gene were designed and tested via transient hairy root transformation, with one (SP1) showing high editing efficiency. Stable transformation generated tomato lines with mutations in SlPG, including insertions, deletions, and null alleles. Homozygous mutant lines exhibited enhanced fruit firmness and delayed softening compared to wild-type, without other morphological differences. Expression analysis found reduced SlPG transcription in mutant lines, demonstrating successful targeted mutagenesis of the SlPG gene using CRISPR
Population Calling: a powerful tool for novel mutation detection in larger sa...GENALICE
This document describes different methods for genotype calling in large population studies, including single sample calling, batch calling, and joint/population calling. It presents a case study comparing the Genalice population calling module to traditional stepwise calling using BWA/GATK on a dataset of 3,000 rice genomes. The population calling approach was over 30 times faster than stepwise calling and scaled linearly with sample size. While it produced fewer total variants than BWA/GATK, over 80% of shared sites had identical genotypes between the two methods. The document concludes that population calling is an efficient approach for large datasets but that more analysis is needed to understand differences from other methods.
A Cost Analysis and Evaluation of Perfused Seed Train Scenarios Through Proce...Merck Life Sciences
Access the interactive recording: https://bit.ly/386d4fh
Abstract:
The bioprocessing industry is driving towards intensified processes to reduce cost of goods and/or increase productivity. Perfused seed is one specific intensified upstream process that can provide benefits to mAb production. To better understand these benefits, BioSolve process modeling software was used to perform a holistic cost analysis of several different perfused seed train scenarios. The effect of variables such as production/seed ratio, number of production bioreactors, titer, and production duration were evaluated. Results showed that under certain scenarios, perfused seed train options could deliver lower cost of goods, increase product throughput, or a combination of both.
A Cost Analysis and Evaluation of Perfused Seed Train Scenarios Through Proce...MilliporeSigma
Access the interactive recording: https://bit.ly/386d4fh
Abstract:
The bioprocessing industry is driving towards intensified processes to reduce cost of goods and/or increase productivity. Perfused seed is one specific intensified upstream process that can provide benefits to mAb production. To better understand these benefits, BioSolve process modeling software was used to perform a holistic cost analysis of several different perfused seed train scenarios. The effect of variables such as production/seed ratio, number of production bioreactors, titer, and production duration were evaluated. Results showed that under certain scenarios, perfused seed train options could deliver lower cost of goods, increase product throughput, or a combination of both.
" Harnessing agricultural biotechnology for resilience to climate change: A l...ExternalEvents
" Harnessing agricultural biotechnology for resilience to
climate change: A lesson from water efficient maize for Africa
project" presentation by Yoseph Beyene, International Maize and Wheat Improvement Center (CIMMYT), Nairobi, Kenya
Speaker: Eduardo Vallejos Associate Professor, Molecular Biology & Physiology.
The talk will cover overall perspective of both genetic and modeling and advanced methods for working with the genetic and phenotypic data with crop models and a perspective on promising future approaches.
Similar to Targeted Breeding Applications of CRISPR-Cas (20)
CoMEHeRe: Co-operative Models for Evidence-based Healthcare RedistributionKate Barlow
CoMEHeRe is a project that aims to give users control over the exploitation of their wearable biosensor data through a blockchain-based infrastructure. It will extract descriptors from raw biosensor data using AI and store hashes of the descriptors on the blockchain to provide provenance. This will allow users to selectively share their data in exchange for incentives from healthcare providers and researchers. An initial prototype was tested with 100 students and showed promising results in extracting stress descriptors. Further trials are planned to refine the socioeconomic models and user experience.
Evaluating How Blockchain Can Transform the Pharmaceutical and Healthcare Ind...Kate Barlow
- Collaborating with 17 companies across the industry
- Understanding the landscape in the pharma and healthcare settings
- Exploring the areas where Blockchain could be used
- Presenting two detailed use cases (a. Smart Contract: Vendor and Site
- Oversight; b. Distributed Asset Ledger: Patient Data Access/Transparency) to support future development and implementation for proof of concept.
NIZO is a contract research organization with expertise in microbiome modulation. They help clients create microbiome modulators through a process of connecting to relevant targets, predicting biological functions, validating functions through various in vitro and in vivo tests, and scaling up production. NIZO has over 70 years of experience in this field and offers a range of services from discovery and validation through to production and clinical trials using their specialized facilities, technologies, and multidisciplinary team of over 130 professionals.
Precision in Plant Immune Expression: Not Lost in Translation Kate Barlow
Xinnian Dong, HHMI Investigator, Arts & Sciences Professor of Biology, Duke University
• Expression of broad-spectrum disease resistance in plants often results in fitness penalty.
• Plant immune responses are normally regulated at both transcriptional and translational levels.
• Transcriptional and translational regulatory mechanisms of plant immune genes can be manipulated to engineer broad-spectrum resistance in crops.
Beyond GWAS QTL Identification and Strategies to Increase YieldKate Barlow
Mohsen Mohammadi, Assistant Professor of Wheat Breeding and Quantitative Genetics, Purdue University
Genetic variation in yield and yield-related traits in an elite population of soft red winter wheat was studied using field-based low-throughput phenotyping and genotyping-by-sequencing markers. QTL conditioning grain yield, grain number per unit area, and kernel weight were identified. QTL result was mined to identify prospects of parents’ complementarity. Strategies for further improvements of grain yield of SRWW populations will be discussed.
Information Technology Meets Synthetic Biology for Ag-Tech Kate Barlow
Until recently, modifying plants to improve traits for improved tolerance and resistance has been a slow and somewhat artisanal process. With advances in recombinant methods and DNA synthesis, systems can now be put in place that vastly accelerate development. In this talk, we will review the progress made and show how systems can be implemented that help automated plant modification R&D.
From Simple to Complex – Phytobiomes and the 2050 Vision for AgricultureKate Barlow
Kellye Eversole, Executive Director, Phytobiomes Alliance & the IWGSC
Phytobiomes are plants in a biome (a specific geophysical environment - e.g., soil, weather -- and all macro- and micro-organisms – e.g., microbes, insects, animals associated with the specific site). Phytobiomes research is a holistic, systems-level approach that integrates many disciplines including geophysics, biology, breeding, agronomy, modeling, and engineering. By focusing on the phytobiome, we will be able to elucidate, quantify, model, predict, act, manipulate, prevent, and ultimately prescribe the cropping systems, methods, and management practices most suited for sustainable production on a particular farm, grassland, or forest. The International Alliance for Phytobiomes Research, an industry-academic consortium, was created in 2016 to bring this vision to fruition. The phytobiomes concept and the research and resource priorities of the Phytobiomes Alliance will be presented.
A Universal Genetic Switch for Increasing Plant Yields, Stress Tolerance and ...Kate Barlow
Jerry Feitelson, Ph.D., Co-Founder & CEO, Agribody Technologies, Inc.
ATI applies a proven solution to food-sourcing problems due to growing populations, extreme weather and decreasing farmland by significantly increasing crop yields. Our patented genetic modification (GM) or genome editing (GE) technology also delays plant senescence, while increasing resistance to diseases and sublethal stresses such as drought, heat, cold, salt, low nutrients and crowding in many key crop plants, including 2 years of field trial data in alfalfa. Many licenses and co-development projects are underway.
Informatics in Context: Managing Sample-to-Answer Multi-Omics WorkflowsKate Barlow
Christopher Mueller, PhD, President & CTO, Lab7 Systems, Inc.
Using a case study from Lab7’s work at the USDA Meat Animal Research Center, we will illustrate how understanding the people, process, and technologies used in a data-intensive mutli-omics laboratory environment is essential to developing comprehensive solutions. Questions that impact the development of a solution include: Who is doing the work? Who is requesting the work? Who needs the results How do samples move through workflows? What types of measurements are needed? What types of data is collected? What analysis is required? What options are available, what options make sense given the people and process? How do the pieces fit together to form a cohesive solution?
Prospects for Digital PCR in Absolute Quantification of DNA and RNAKate Barlow
Ward De Spiegelaere, Assistant Professor, Ghent University, Belgium
• Using examples of virology I will talk about the possibilities
and pitfalls of current digital PCR technology for absolute
quantification of DNA and RNA
• I will expand on a tool we developed for data driven
threshold setting in digital PCR
• I will discuss a method for normalization of RNA data in
digital PCR
Challenges and Opportunities for Digital PCR in the CLIA Laboratory of the Mo...Kate Barlow
Anthony Magliocco, Chair of Anatomical Pathology, Moffitt Cancer Center, USA
The Moffit Cancer Center is one of the largest NCI designated comprehensive free-standing cancer centers in the USA. The center has developed one of the most advanced personalized cancer medicine treatment programs in the world. This program is supported by a comprehensive and advanced CLIA molecular diagnostics. Digital PCR assays are currently being developed for several clinical applications including TKI resistance monitoring in patients with advanced lung cancer. The challenges and opportunities in deploying digital PCR into clinical practice will be discussed.
Circulating Tumor DNA Detection from Heparinized Plasma Samples by Droplet Di...Kate Barlow
Nasrin Sarafan-Vasseur, Liquid Biopsy Scientific Leader, Research Team on Oncology (IRON), INSERM and University of Rouen, France
Heparin is often used as plasma anticoagulant for tumor marker analysis but corresponds also to an inhibitory of PCR not enabling circulating tumor DNA (ctDNA) detection, which has been highlighted as a potential “liquid biopsy”. We evaluated the impact of heparinase addition on heparinized plasma samples to allow ctDNA analysis. Circulating ESR1 and KRAS mutations were quantified by digital PCR, in plasma collected in heparinized tubes (n=194) from hormone receptor-positive metastatic breast cancer (HR+MBC) and pancreatic adenocarcinoma (PA) patients. We improved significantly PCR efficiency in 91/144 HR+MBC and 26/50 PA plasma samples, enabling ctDNA detection in 22/91 and 13/26 patients. This new processing did not alter quantitatively and qualitatively ctDNA detection and could make the samples from heparinized blood-derived collections suitable for ctDNA analysis.
Two-Tailed PCR - New Ultrasensitive and Ultraspecific Technique for the Quant...Kate Barlow
This document discusses methods for quantifying and analyzing microRNAs (miRNAs) using quantitative PCR (qPCR). It presents a new two-tailed RT-qPCR method that provides high sensitivity and specificity for detecting miRNAs, including discrimination of miRNA isoforms. The method allows unlimited multiplexing in the reverse transcription step followed by singleplex qPCR. The document benchmarks the two-tailed RT-qPCR method on biological samples, showing it can sensitively detect less than 10 molecules and maintain specificity across the entire miRNA sequence. It also demonstrates two-tube multiplexing of the method to profile expression levels of several miRNAs in different tissues.
Admix™: Custom lyophilised RT-PCR reagents for point-of-use applicationsKate Barlow
This document discusses various enzyme products including low glycerol enzymes, no glycerol enzymes, and lyo ready enzymes and buffer kits. It also mentions solid A + B and lists features of various enzyme products. Additionally, the document outlines a typical product development process involving specification, formulation, first samples, finished R&D verification, process validation, supply chain setup, and manufacture.
Explaining Biocide Tolerance of Gram Negative Bacteria Kate Barlow
Working on multiple organisms and constantly changing gene-targets requires use of an easily optimisable and cheap qPCR method, which is why we use SyBr Green qPCR. We have investigated chlorhexidine resistance in Klebsiella pneumoniae and are about to publish on biocide resistance in Acinetobacter baumannii and Pseudomonas aeruginosa. I will explain our approach and our optimisation and robustness strategies, as well as an overview of the hypotheses we developed and confirmed using our qPCR approach.
Lucy Bock, Senior Scientist/Project Team Leader, Technology Development Group, Public Health England, UK
Optimized Design of Broadly Detecting qPCR Primers and Probes Using a Conserv...Kate Barlow
Jonas Blomberg, Emeritus Professor of Clinical Virology, Uppsala University, Sweden
Design of broadly detecting qPCRs is a challenge. It requires both an accurate analysis of sequence conservation and of how primers and probes interact with their targets. Hybridization prediction has gone from a simple reliance on GC content to more precise algorithms. Nearest neighbour analysis relies on short distance prediction. We developed an algorithm for longer nucleotide distances, NucZip. It allows design of long primers and probes, using wobble positions and inosine. A computer program which embodies both conservation analysis and hybridization prediction, ConSort, was developed. We used it for development of qPCRs for Orthomyxo-, Corona-, Entero-, Retro- and Noroviruses. Different aspects of the design process will be discussed.
MelTree: A Novel Workflow for the Automated Identification of a Large Number ...Kate Barlow
Jean-Christophe Avarre, Head of the High Throughput qPCR Platform and Research Group Leader, University of Montpellier, France
Nucleic acid characterization by High Resolution Melting (HRM) is a simple, flexible, low-cost and powerful technique for identifying sequence variations, making it attractive for a broad range of diagnostic and research applications. However, current procedures for analyzing HRM curves are not suited for large data sets.
For this reason, we have developed an innovative method that enables the simultaneous discrimination of a large number of variants from their HRM profile. This method relies on the establishment of a melting profile library and offers a fully automated analysis workflow.
Using this workflow, it was possible to discriminate 19 nontuberculous mycobacterial species from their HRM profile. It was also possible to design a multiplex diagnostic tool targeting five pathogens responsible for abortive diseases in cattle.
Discordance Between Replicate qPCR ReactionsKate Barlow
In the analysis of qPCR data, the Cq values of replicate reactions is required to differ no more than 0.5 cycles. However, the sampling error that occurs when pipetting a low number of target molecules into the PCR plate is governed by the Poisson distribution. For each Cq and PCR efficiency value we calculated this unavoidable range of Cq values. This range increases with higher Cq values (less target). A decision to exclude replicate reactions based on this expected sampling error avoids bias, prevents unwanted loss of data and increases the statistical power. For a dataset with replicate qPCR measurements of 12 miRNA targets in 834 patients (20,016 reactions) the fraction of excluded measurements decreased from 39% to 7%.In the analysis of qPCR data, the Cq values of replicate reactions is required to differ no more than 0.5 cycles. However, the sampling error that occurs when pipetting a low number of target molecules into the PCR plate is governed by the Poisson distribution. For each Cq and PCR efficiency value we calculated this unavoidable range of Cq values. This range increases with higher Cq values (less target). A decision to exclude replicate reactions based on this expected sampling error avoids bias, prevents unwanted loss of data and increases the statistical power. For a dataset with replicate qPCR measurements of 12 miRNA targets in 834 patients (20,016 reactions) the fraction of excluded measurements decreased from 39% to 7%.
Jan Ruijter, Assistant Professor, University of Amsterdam, The Netherlands
Development and Clinical Validation of Liquid ddPCR Tests for Actionable Soma...Kate Barlow
We have developed and validated blood-based variant specific ddPCR tests for EGFR, KRAS, BRAF, EML4-ALK, ROS1 and RET variants. These tests are intended for use in patients diagnosed with Non-Small Cell Lung Cancer (NSCLC). The tests have been on the market as ”the GeneStrat® test” since 2015; and in that time, has been utilized to analyze over 80,000 individual variants. Greater than 90% of tests have been delivered in less than 72 hours from receipt at the testing Laboratory. We will report on factors critical to the development, validation and on-market support of these tests. In this talk we will cover:
• Learning how Droplet Digital ™ PCR technology is being used for liquid biopsy testing in the clinical setting
• Reviewing development and validation case studies for cfDNA testing using ddPCR
• Reviewing performance data and quality metrics from on-market experiences
Gary Pestano, Vice President, Development and Operations, Biodesix
Clinical Utility of Droplet Digital PCR on Liquid Biopsies from Patients with...Kate Barlow
The Treatment Resistance Team at the Institute of Cancer Research has been using plasma to interrogate resistance in castration-resistant prostate cancer (CRPC) and develop biomarkers for selecting treatment. Using targeted next-generation sequencing and droplet digital PCR on cfDNA from sequential plasma samples AR mutations was found to emerge with resistance to abiraterone and enzalutamide. A strong association between plasma AR aberrations in the form of AR gain and mutations and resistance to abiraterone or enzalutamide in CRPC patients was also seen, supporting the clinical utility of cfDNA studies in metastatic prostate cancer.
Daniel Wetterskog, Senior Scientist, Institute of Cancer Research, UK
Detecting visual-media-borne disinformation: a summary of latest advances at ...VasileiosMezaris
We present very briefly some of the most important and latest (June 2024) advances in detecting visual-media-borne disinformation, based on the research work carried out at the Intelligent Digital Transformation Laboratory (IDT Lab) of CERTH-ITI.
Cultivation of human viruses and its different techniques.MDAsifKilledar
Viruses are extremely small, infectious agents that invade cells of all types. These have been culprits in many human disease including small pox,flu,AIDS and ever present common cold as well as plants bacteria and archea .
Viruses cannot multiply outside the living host cell, However the isolation, enumeration and identification become a difficult task. Instead of chemical medium they require a host body.
Viruses can be cultured in the animals such as mice ,monkeys, rabbits and guinea pigs etc. After inoculation animals are carefully examined for the development of signs or symptoms, further they may be killed.
Discovery of Merging Twin Quasars at z=6.05Sérgio Sacani
We report the discovery of two quasars at a redshift of z = 6.05 in the process of merging. They were
serendipitously discovered from the deep multiband imaging data collected by the Hyper Suprime-Cam (HSC)
Subaru Strategic Program survey. The quasars, HSC J121503.42−014858.7 (C1) and HSC J121503.55−014859.3
(C2), both have luminous (>1043 erg s−1
) Lyα emission with a clear broad component (full width at half
maximum >1000 km s−1
). The rest-frame ultraviolet (UV) absolute magnitudes are M1450 = − 23.106 ± 0.017
(C1) and −22.662 ± 0.024 (C2). Our crude estimates of the black hole masses provide log 8.1 0. ( ) M M BH = 3
in both sources. The two quasars are separated by 12 kpc in projected proper distance, bridged by a structure in the
rest-UV light suggesting that they are undergoing a merger. This pair is one of the most distant merging quasars
reported to date, providing crucial insight into galaxy and black hole build-up in the hierarchical structure
formation scenario. A companion paper will present the gas and dust properties captured by Atacama Large
Millimeter/submillimeter Array observations, which provide additional evidence for and detailed measurements of
the merger, and also demonstrate that the two sources are not gravitationally lensed images of a single quasar.
Unified Astronomy Thesaurus concepts: Double quasars (406); Quasars (1319); Reionization (1383); High-redshift
galaxies (734); Active galactic nuclei (16); Galaxy mergers (608); Supermassive black holes (1663)
Compositions of iron-meteorite parent bodies constrainthe structure of the pr...Sérgio Sacani
Magmatic iron-meteorite parent bodies are the earliest planetesimals in the Solar System,and they preserve information about conditions and planet-forming processes in thesolar nebula. In this study, we include comprehensive elemental compositions andfractional-crystallization modeling for iron meteorites from the cores of five differenti-ated asteroids from the inner Solar System. Together with previous results of metalliccores from the outer Solar System, we conclude that asteroidal cores from the outerSolar System have smaller sizes, elevated siderophile-element abundances, and simplercrystallization processes than those from the inner Solar System. These differences arerelated to the formation locations of the parent asteroids because the solar protoplane-tary disk varied in redox conditions, elemental distributions, and dynamics at differentheliocentric distances. Using highly siderophile-element data from iron meteorites, wereconstruct the distribution of calcium-aluminum-rich inclusions (CAIs) across theprotoplanetary disk within the first million years of Solar-System history. CAIs, the firstsolids to condense in the Solar System, formed close to the Sun. They were, however,concentrated within the outer disk and depleted within the inner disk. Future modelsof the structure and evolution of the protoplanetary disk should account for this dis-tribution pattern of CAIs.
Signatures of wave erosion in Titan’s coastsSérgio Sacani
The shorelines of Titan’s hydrocarbon seas trace flooded erosional landforms such as river valleys; however, it isunclear whether coastal erosion has subsequently altered these shorelines. Spacecraft observations and theo-retical models suggest that wind may cause waves to form on Titan’s seas, potentially driving coastal erosion,but the observational evidence of waves is indirect, and the processes affecting shoreline evolution on Titanremain unknown. No widely accepted framework exists for using shoreline morphology to quantitatively dis-cern coastal erosion mechanisms, even on Earth, where the dominant mechanisms are known. We combinelandscape evolution models with measurements of shoreline shape on Earth to characterize how differentcoastal erosion mechanisms affect shoreline morphology. Applying this framework to Titan, we find that theshorelines of Titan’s seas are most consistent with flooded landscapes that subsequently have been eroded bywaves, rather than a uniform erosional process or no coastal erosion, particularly if wave growth saturates atfetch lengths of tens of kilometers.
Mapping the Growth of Supermassive Black Holes as a Function of Galaxy Stella...Sérgio Sacani
The growth of supermassive black holes is strongly linked to their galaxies. It has been shown that the population
mean black hole accretion rate (BHAR) primarily correlates with the galaxy stellar mass (Må) and redshift for the
general galaxy population. This work aims to provide the best measurements of BHAR as a function of Må and
redshift over ranges of 109.5 < Må < 1012 Me and z < 4. We compile an unprecedentedly large sample with 8000
active galactic nuclei (AGNs) and 1.3 million normal galaxies from nine high-quality survey fields following a
wedding cake design. We further develop a semiparametric Bayesian method that can reasonably estimate BHAR
and the corresponding uncertainties, even for sparsely populated regions in the parameter space. BHAR is
constrained by X-ray surveys sampling the AGN accretion power and UV-to-infrared multiwavelength surveys
sampling the galaxy population. Our results can independently predict the X-ray luminosity function (XLF) from
the galaxy stellar mass function (SMF), and the prediction is consistent with the observed XLF. We also try adding
external constraints from the observed SMF and XLF. We further measure BHAR for star-forming and quiescent
galaxies and show that star-forming BHAR is generally larger than or at least comparable to the quiescent BHAR.
Unified Astronomy Thesaurus concepts: Supermassive black holes (1663); X-ray active galactic nuclei (2035);
Galaxies (573)
Continuing with the partner Introduction, Tampere University has another group operating at the INSIGHT project! Meet members of the Industrial Engineering and Management Unit - Aki, Jaakko, Olga, and Vilma!
SAP Unveils Generative AI Innovations at Annual Sapphire ConferenceCGB SOLUTIONS
At its annual SAP Sapphire conference, SAP introduced groundbreaking generative AI advancements and strategic partnerships, underscoring its commitment to revolutionizing business operations in the AI era. By integrating Business AI throughout its enterprise cloud portfolio, which supports the world's most critical processes, SAP is fostering a new wave of business insight and creativity.
Embracing Deep Variability For Reproducibility and Replicability
Abstract: Reproducibility (aka determinism in some cases) constitutes a fundamental aspect in various fields of computer science, such as floating-point computations in numerical analysis and simulation, concurrency models in parallelism, reproducible builds for third parties integration and packaging, and containerization for execution environments. These concepts, while pervasive across diverse concerns, often exhibit intricate inter-dependencies, making it challenging to achieve a comprehensive understanding. In this short and vision paper we delve into the application of software engineering techniques, specifically variability management, to systematically identify and explicit points of variability that may give rise to reproducibility issues (eg language, libraries, compiler, virtual machine, OS, environment variables, etc). The primary objectives are: i) gaining insights into the variability layers and their possible interactions, ii) capturing and documenting configurations for the sake of reproducibility, and iii) exploring diverse configurations to replicate, and hence validate and ensure the robustness of results. By adopting these methodologies, we aim to address the complexities associated with reproducibility and replicability in modern software systems and environments, facilitating a more comprehensive and nuanced perspective on these critical aspects.
https://hal.science/hal-04582287
Dr. Firoozeh Kashani-Sabet is an innovator in Middle Eastern Studies and approaches her work, particularly focused on Iran, with a depth and commitment that has resulted in multiple book publications. She is notable for her work with the University of Pennsylvania, where she serves as the Walter H. Annenberg Professor of History.
2. • Why we are excited about genome editing
• Genome editing technology
• Examples
3. Six vegetables that are the same biological species
Kohlrabi Kale Broccoli Brussels Sprouts Cabbage Cauliflower
Wild mustard
Genome editing replicates naturally occurring outcomes
4. Duplication of SUN gene
changes fruit morphology
Genome editing replicates naturally occurring outcomes
5. Between two corn
varieties
• 100s of genes with
different numbers of
copies
• 1,000s of genes
absent in one but
present in the other
Genome editing replicates naturally occurring outcomes
6. Genome editing replicates naturally occurring outcomes
• 7 single nucleotide
mutations per billion
base pairs per
generation
• 1 hectare of soy has
>1.8 million novel
mutations
7. Year: 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Editing
Edited variety development can be rapid
Breeding
Transgenes
1st commercial
sales in year 13-20
1st commercial
sales in year 4
1st commercial
sales in year 9
15. Vectors containing Babyboom and Wuschel trigger
rapid somatic embryogenesis
Embryos at
7 days
Well
formed
somatic
embryos
visible at 2-
4 days
High
mitotic
index in
young
embryos
15
16. Site-Seq can identify off-sites
High molecular weight
genomic DNA
Digest with Cas9 RNP
“A” addition
Ligate biotinylated adapter
Random shear / size select
“ A” addition, ligation to adapters
Biotin selection
PCR
Sequencing
17. 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 PAM
G G C C G A G G T C G A C T A C C G G C CGG M2 MS45-CR2
C G C C G A G G G C G A C T A C C G G C AGG Off-Site 1
G G T G G G G G T C G A C T A C C G G C AGG Off-Site 2
C G A C T A G A T C G A C T A C C G G C GGG Off-Site 3
C G C C G C G G G C G A C T A C C G G C AGG Off-Site 4
G G G A C G A G G C G A T T A C C G G C GGG Off-Site 5
A G A C G A T G T C G A C C G C C G C GGG Off-Site 6
G C C C C A T G T C G A C T A C C G G C AGG Off-Site 7
A T G G C T A G G A G A C T A C C G G C TGG Off-Site 8
Red = Mismatch; Black = Gap
Site-Seq can identify off-sites
18. 1. Select unique sites informatically
Achieving specificity through guide design and testing
1 Mismatch 2 Mismatch 3 Mismatch 4 Mismatch
Site 1 0 0 0 0
Site 2 0 0 0 0
Site 3 0 0 0 23
Site 4 0 0 8 34
Site 5 0 0 10 41
Site 6 0 0 54 261
Site 7 0 1 51 236
Site 8 0 2 151 116
Site 9 0 3 48 126
Site 10 0 7 117 396
Site 11 0 9 96 528
Site 12 0 10 317 2209
Site 13 0 32 674 1142
Site 14 1 19 764 2713
Site 15 3 41 433 635
Number of Predicted Off-Sites
Effect of RNP Concentration on On- and Off-Site Cleavage
20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 PAM 10 nM 100 nM 1000 nM
On-Site G G C C G A G G T C G A C T A C C G G C CGG Y Y Y
Off-Site 1 C G C C G A G G G C G A C T A C C G G C AGG Y Y Y
Off-Site 2 G G T G G G G G T C G A C T A C C G G C AGG Y Y Y
Off-Site 3 C G A C T A G A T C G A C T A C C G G C GGG N Y Y
Off-Site 4 C G C C G C G G G C G A C T A C C G G C AGG N Y Y
Off-Site 5 G G G A C G A G G C G A T T A C C G G C GGG N N Y
Protospacer RNP Concentration
2. Confirm target specificity in vitro (SITE-Seq+)
25. Status
• Edits made in multiple inbreds – can be
combined to make 10 elite hybrids
• Contain expected edit; no other DNA
• Expected waxy seed composition
Wild-type Wx1
Transcriptional start
Guide 1 Guide 2
4kb deletion
Edited Wx1
Iodine stain of segregating
seeds
Waxy
kernel
Stains
lighter
Waxy corn gene editing approach: gene deletion
26. Regulatory/ Acceptance
CRISPR waxy large scale field trials summer 2017
• USDA: not regulated by its Biotechnology Regulatory Service
• CONABIA: not a “GMO” under Argentina law
• Other import markets: discussions underway
Wild type Conventional waxy CRISPR waxy
27. Example #2: corn northern leaf blight (NLB)
Fungal disease caused by Exserohilum turcicum Grain yield loss can be 18% - 98%
28. Resistance to northern leaf blight
• NLB18 gene cloned and encodes
a receptor-like kinase
• Transgenic maize plants
overexpressing NLB18 with
heterologous promoter are
resistant to NLB
NLB18 overexpression Wild type
29. Inserted the resistant NLB18 gene at a
breeding stack locus
NLB gene editing approach
(a): gene insertion
Hemizygous Null Homozygous
1.0
1.8
NLB 18 expression
RT-PCR
2017 field testing
5.4
v
v
v
Chr1
Resistant
allele
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