scholarly journals Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis

2021 ◽  
Vol 22 (22) ◽  
pp. 12611
Author(s):  
Susan Schröpfer ◽  
Henryk Flachowsky
Keyword(s):  
Genome Editing ◽  
High Throughput ◽  
Gel Electrophoresis ◽  
Cost Effective ◽  
Capillary Gel Electrophoresis ◽  
Guide Rnas ◽  
Large Deletions ◽  
Multiple Loci ◽  
Fluorescent Pcr

The use of the novel CRISPR/Cas12a system is advantageous, as it expands the possibilities for genome editing (GE) applications due to its different features compared to the commonly used CRISPR/Cas9 system. In this work, the CRISPR/Cas12a system was applied for the first time to apple to investigate its general usability for GE applications. Efficient guide RNAs targeting different exons of the endogenous reporter gene MdPDS, whose disruption leads to the albino phenotype, were pre-selected by in vitro cleavage assays. A construct was transferred to apple encoding for a CRISPR/Cas12a system that simultaneously targets two loci in MdPDS. Using fluorescent PCR capillary electrophoresis and amplicon deep sequencing, all identified GE events of regenerated albino shoots were characterized as deletions. Large deletions between the two neighboring target sites were not observed. Furthermore, a chimeric composition of regenerates and shoots that exhibited multiple GE events was observed frequently. By comparing both analytical methods, it was shown that fluorescent PCR capillary gel electrophoresis is a sensitive high-throughput genotyping method that allows accurate predictions of the size and proportion of indel mutations for multiple loci simultaneously. Especially for species exhibiting high frequencies of chimerism, it can be recommended as a cost-effective method for efficient selection of homohistont GE lines.

scholarly journals High-throughput genotyping of CRISPR/Cas9-mediated mutants using fluorescent PCR-capillary gel electrophoresis

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Muhammad Khairul Ramlee ◽  
Tingdong Yan ◽  
Alice M. S. Cheung ◽  
Charles T. H. Chuah ◽  
Shang Li
Keyword(s):  
High Throughput ◽  
Gel Electrophoresis ◽  
Capillary Gel Electrophoresis ◽  
Fluorescent Pcr

scholarly journals 230 Genome editing applications in plants: high-throughput CRISPR/Cas editing for crop improvement

2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 56-56
Author(s):  
Michael Thomson
Keyword(s):  
Genome Editing ◽  
High Throughput ◽  
Positional Cloning ◽  
Gene Editing ◽  
Crop Improvement ◽  
Cost Effective ◽  
Gene Families ◽  
Ease Of Use ◽  
Plant Genome ◽  
Wide Range

Abstract The precision and ease of use of CRISPR nucleases, such as Cas9 and Cpf1, for plant genome editing has the potential to accelerate a wide range of applications for crop improvement. For upstream research on gene discovery and validation, rapid gene knock-outs can enable testing of single genes and multi-gene families for functional effects. Large chromosomal deletions can test the function of tandem gene arrays and assist with positional cloning of QTLs by helping to narrow down the target region. Nuclease-deactivated Cas9 fusion proteins with transcriptional activators and repressors can be used to up and down-regulate gene expression. Even more promising, gene insertions and allele replacements can provide the opportunity to rapidly test the effects of different alleles at key loci in the same genetic background, providing a more precise alternative to marker-assisted backcrossing. Recently, Texas A&M AgriLife Research has supported the development of a Crop Genome Editing Lab at Texas A&M working towards optimizing a high-throughput gene editing pipeline and providing an efficient and cost-effective gene editing service for research and breeding groups. The lab is using rice as a model to test and optimize new approaches aimed towards overcoming current bottlenecks. For example, a wealth of genomics data from the rice community enables the development of novel approaches to predict which genes and target modifications may be most beneficial for crop improvement, taking advantage of known major genes, high-resolution GWAS data, multiple high-quality reference genomes, transcriptomics data, and resequencing data from the 3,000 Rice Genomes Project. Current projects have now expanded to work across multiple crops to provide breeding and research groups with a rapid gene editing pipeline to test candidate genes in their programs, with the ultimate goal of developing nutritious, high-yielding, stress-tolerant crops for the future.

scholarly journals High-throughput glycosylation analysis of therapeutic immunoglobulin G by capillary gel electrophoresis using a DNA analyzer

mAbs ◽  
2013 ◽  
Vol 6 (1) ◽  
pp. 185-196 ◽  
Author(s):  
Dietmar Reusch ◽  
Markus Haberger ◽  
Tobias Kailich ◽  
Anna-Katharina Heidenreich ◽  
Michael Kampe ◽  
...  
Keyword(s):  
High Throughput ◽  
Immunoglobulin G ◽  
Gel Electrophoresis ◽  
Capillary Gel Electrophoresis

scholarly journals Amyloid fibril-based hydrogels for high-throughput tumor spheroid modeling

2020 ◽  
Author(s):  
Namrata Singh ◽  
Komal Patel ◽  
Ambuja Navalkar ◽  
Pradeep Kadu ◽  
Debalina Datta ◽  
...  
Keyword(s):  
Drug Resistance ◽  
High Throughput ◽  
Three Dimensional ◽  
3D Cell Culture ◽  
Cancer Therapeutics ◽  
Cost Effective ◽  
Cell Types ◽  
Tumor Spheroid ◽  
Tumor Modeling

AbstractBiomaterials mimicking extracellular matrices (ECM) for three-dimensional (3D) cultures have gained immense interest in tumor modeling and in vitro organ development. Here, we introduce versatile, thixotropic amyloid hydrogels as a bio-mimetic ECM scaffold for 3D cell culture as well as high-throughput tumor spheroid formation using a drop cast method. The unique cross-β-sheet structure, sticky surface, and thixotropicity of amyloid hydrogels allow robust cell adhesion, survival, proliferation, and migration, which are essential for 3D tumor modeling with various cancer cell types. The spheroids formed show overexpression of the signature cancer biomarkers and confer higher drug resistance compared to two-dimensional (2D) monolayer cultures. Using breast tumor tissue from mouse xenograft, we showed that these hydrogels support the formation of tumor spheroids with a well-defined necrotic core, cancer-associated gene expression, higher drug resistance, and tumor heterogeneity reminiscent of the original tumor. Altogether, we have developed a rapid and cost-effective platform for generating in vitro cancer models for the screening of anti-cancer therapeutics and developing personalized medicines.

Cell-based models for mycotoxin screening and toxicity evaluation: an update

2014 ◽  
Vol 7 (2) ◽  
pp. 153-166 ◽  
Author(s):  
F. Cheli ◽  
E. Fusi ◽  
A. Baldi
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Mycotoxin Research ◽  
Cost Effective ◽  
Screening Tests ◽  
Test Model ◽  
Related Factors ◽  
Technical Problems ◽  
Third Dimension

This review presents the applications of cell-based models in mycotoxin research, with a focus on models for mycotoxin screening and cytotoxicity evaluation. Various cell-based models, cell and cell culture condition related factors, toxicity endpoints and culture systems as well as predictive value of cell-based bioassays are reviewed. Advantages, drawbacks and technical problems regarding set up and validation of consistent, robust, reproducible and high-throughput cell-based models are discussed. Various cell-based models have been developed and used as screening tests for mycotoxins but the data obtained are difficult to compare. However, the results highlight the potential of cell-based models as promising in vitro platforms for the initial screening and cytotoxicity evaluation of mycotoxins and as a significant analytical approach in mycotoxin research before any animal or human clinical studies. To develop cell-based models as powerful high-throughput laboratory platforms for the analysis of large numbers of samples, there are mainly two fundamental requirements that should be met, i.e. the availability of easy-to-use and, if possible, automated cell platforms and the possibility to obtain reproducible results that are comparable between laboratories. The transition from a research model to a test model still needs optimisation, standardisation, and validation of analytical protocols. The validation of a cell-based bioassay is a complex process, as several critical points, such as the choice of the cellular model, the assay procedures, and the appropriate use and interpretation of the results, must be strictly defined to ensure more consistency in the results. The development of cell-based models exploring the third dimension together with automation and miniaturisation will bring cellular platforms to a level appropriate for cost-effective and large-scale analysis in the field of mycotoxin research.

scholarly journals High-throughput in vitro specificity profiling of natural and high-fidelity CRISPR-Cas9 variants

2020 ◽  
Author(s):  
Karthik Murugan ◽  
Arun S. Seetharam ◽  
Andrew J. Severin ◽  
Dipali G. Sashital
Keyword(s):  
Genome Editing ◽  
High Throughput ◽  
Double Strand Breaks ◽  
Target Sequence ◽  
Wild Type ◽  
Strand Breaks ◽  
Cleavage Assay ◽  
Target Sequences

AbstractCas9 is an RNA-guided endonuclease in the bacterial CRISPR-Cas immune system and a popular tool for genome editing. The most commonly used Cas9 variant, Streptococcus pyogenes Cas9 (SpCas9), is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific than wild-type (WT) SpCas9. However, systematic comparisons of the cleavage activities of these Cas9 variants have not been reported. In this study, we employed our high-throughput in vitro cleavage assay to compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9, and HiFi Cas9). We observed that all Cas9s tested were able to cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on the target sequence and Cas9 variant. For targets with multiple mismatches, SaCas9 and engineered SpCas9 variants are more prone to nicking, while WT SpCas9 creates double-strand breaks (DSB). These differences in cleavage rates and DSB formation may account for the varied specificities observed in genome editing studies. Our analysis reveals mismatch position-dependent, off-target nicking activity of Cas9 variants which have been underreported in previous in vivo studies.

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