scholarly journals A simple and reliable method for creating PCR-detectable mutants in Arabidopsis with the polycistronic tRNA–gRNA CRISPR/Cas9 system

2019 ◽  
Vol 41 (10) ◽  
Author(s):  
Liangliang Hui ◽  
Min Zhao ◽  
Junqi He ◽  
Yingying Hu ◽  
Yanshuang Huo ◽  
...  
Keyword(s):  
Gene Editing ◽  
Reliable Method ◽  
Screening Methods ◽  
Deletion Mutants ◽  
Pcr Screening ◽  
Large Deletions ◽  
System A ◽  
Homozygous Mutant ◽  
Laborious Work ◽  
Rapid Tool

Abstract To develop an easy and robust method for creating genetically stable and easily detectable Arabidopsis mutants, we adopted the polycistronic tRNA–gRNA CRISPR/Cas9 (PTG/Cas9) system, a multiplex gene-editing tool in rice, with PTOX as the reporter gene. The PTG/Cas9 system has a great potential in generating large deletions detectable by PCR, which greatly simplifies the laborious work of mutant screening. We constructed a PTOX–PTG/Cas9 system with five gRNAs and introduced it into Arabidopsis. At T1 generation, 24.4% of transgenic plants were chimeric with PCR-detectable deletions in PTOX locus, but no homozygous mutant was found, indicating that gene editing occurred predominantly in somatic cells. After a self-cross propagation, 60% of T1 chimeric plants were able to produce homozygous, heterozygous, or bi-allelic ptox offsprings. Inheritable homozygous ptox mutants without Cas9 gene can be obtained earliest at T2 generation. We further targeted five other genes using the same procedure and achieved homozygous Cas9-free mutants with large deletions for all genes within three generations. We established a standard and reliable protocol to generate stable inherited deletion mutants in 2–3 generations along with simple PCR screening methods. We conclude that the rice PTG/Cas9 system is an efficient, easy, and rapid tool to edit genes in Arabidopsis. We propose that it could be applied to other genes in Arabidopsis, and it might have the potential to edit genes in other plant species as well.

scholarly journals Detection of Exon Deletions within an Entire Gene (CFTR) by Relative Quantification on the LightCycler

2006 ◽  
Vol 52 (11) ◽  
pp. 2005-2012 ◽  
Author(s):  
Mircea Schneider ◽  
Franziska Joncourt ◽  
Javier Sanz ◽  
Thomas von Känel ◽  
Sabina Gallati
Keyword(s):  
Gene Dosage ◽  
Large Deletion ◽  
Rapid Screening ◽  
Cftr Gene ◽  
Screening Methods ◽  
Sequence Variant ◽  
Β2 Microglobulin ◽  
Large Deletions ◽  
Specific Deletion ◽  
Entire Gene

Abstract Background: Cystic fibrosis (CF) is associated with at least 1 pathogen point sequence variant on each CFTR allele. Some symptomatic patients, however, have only 1 detectable pathogen sequence variant and carry, on the other allele, a large deletion that is not detected by conventional screening methods. Methods: For relative quantitative real-time PCR detection of large deletions in the CFTR gene, we designed DNA-specific primers for each exon of the gene and primers for a reference gene (β2-microglobulin). For PCR we used a LightCycler system (Roche) and calculated the gene-dosage ratio of CFTR to β2-microglobulin. We tested the method by screening all 27 exons in 3 healthy individuals and 2 patients with only 1 pathogen sequence variant. We then performed specific deletion screenings in 10 CF patients with known large deletions and a blinded analysis in which we screened 24 individuals for large deletions by testing 8 of 27 exons. Results: None of the ratios for control samples were false positive (for deletions or duplications); moreover, for all samples from patients with known large deletions, the calculated ratios for deleted exons were close to 0.5. In addition, the results from the blinded analysis demonstrated that our method can also be used for the screening of single individuals. Conclusions: The LightCycler assay allows reliable and rapid screening for large deletions in the CFTR gene and detects the copy number of all 27 exons.

scholarly journals Design of a Surgical Port for Minimally Invasive Beating-Heart Intracardial Procedures

2011 ◽  
Vol 5 (4) ◽  
Author(s):  
Christopher M. DiBiasio ◽  
Keith V. Durand ◽  
Jonathan Hopkins ◽  
Zach Traina ◽  
Alexander H. Slocum ◽  
...  
Keyword(s):  
Minimally Invasive ◽  
Surgical Procedures ◽  
Beating Heart ◽  
Surgical Instruments ◽  
Direct Access ◽  
Surgical Instrument ◽  
System A ◽  
Pulmonary Bypass ◽  
Heart Wall ◽  
Rapid Tool

Direct-access, minimally invasive, beating-heart intracardial procedures have the potential to replace many traditional surgical procedures requiring cardio-pulmonary bypass as long as micro-emboli are prevented from entering the cardiovascular system. A new surgical port was developed to introduce surgical instruments into chambers of the beating heart during minimally invasive, intracardial surgical procedures without allowing the introduction of micro-emboli 0.1 mm or larger in size. The design consists of an outer port body that is secured to the heart wall using a purse string suture and a series of inner tubular sleeves that form the interface between the port and the transecting instrument. The design enables rapid tool changes and accommodates a wide variety of instruments. The port uses a fluid purging system to dislodge and remove emboli from a surgical instrument. Laboratory and clinical tests show that the port adequately seals around a surgical instrument and prevents the introduction of emboli with diameters greater than 0.1 mm into the heart while minimizing hemorrhage.

scholarly journals Current Advancements and Limitations of Gene Editing in Orphan Crops

2021 ◽  
Vol 12 ◽  
Author(s):  
Matthew Venezia ◽  
Kate M. Creasey Krainer
Keyword(s):  
Gene Editing ◽  
Crop Improvement ◽  
Screening Methods ◽  
In Planta ◽  
Base Editing ◽  
Guide Rna ◽  
Orphan Crops ◽  
Associated Proteins ◽  
Improvement Programs ◽  
Novel Alleles

Gene editing provides precise, heritable genome mutagenesis without permanent transgenesis, and has been widely demonstrated and applied in planta. In the past decade, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) has revolutionized the application of gene editing in crops, with mechanistic advances expanding its potential, including prime editing and base editing. To date, CRISPR/Cas has been utilized in over a dozen orphan crops with diverse genetic backgrounds, leading to novel alleles and beneficial phenotypes for breeders, growers, and consumers. In conjunction with the adoption of science-based regulatory practices, there is potential for CRISPR/Cas-mediated gene editing in orphan crop improvement programs to solve a plethora of agricultural problems, especially impacting developing countries. Genome sequencing has progressed, becoming more affordable and applicable to orphan crops. Open-access resources allow for target gene identification and guide RNA (gRNA) design and evaluation, with modular cloning systems and enzyme screening methods providing experimental feasibility. While the genomic and mechanistic limitations are being overcome, crop transformation and regeneration continue to be the bottleneck for gene editing applications. International collaboration between all stakeholders involved in crop improvement is vital to provide equitable access and bridge the scientific gap between the world’s most economically important crops and the most under-researched crops. This review describes the mechanisms and workflow of CRISPR/Cas in planta and addresses the challenges, current applications, and future prospects in orphan crops.

scholarly journals Novel TaqMan PCR screening methods for element cry3A and construct gat/T-pinII to support detection of both known and unknown GMOs

2016 ◽  
Vol 243 (3) ◽  
pp. 481-488 ◽  
Author(s):  
Theo W. Prins ◽  
Richard A. van Hoof ◽  
Ingrid M. J. Scholtens ◽  
Esther J. Kok
Keyword(s):  
Screening Methods ◽  
Pcr Screening ◽  
Taqman Pcr

Dietary changes improve survival of CFTR S489X homozygous mutant mouse

1995 ◽  
Vol 269 (5) ◽  
pp. L625-L630 ◽  
Author(s):  
E. A. Eckman ◽  
C. U. Cotton ◽  
D. M. Kube ◽  
P. B. Davis
Keyword(s):  
Growth Rate ◽  
Reliable Method ◽  
Mutant Mouse ◽  
Liquid Diet ◽  
Pathological Changes ◽  
Corn Cob ◽  
Dietary Changes ◽  
Homozygous Mutant ◽  
Homozygous Mutant Mouse ◽  
Survival And Growth

Over 90% of untreated CFTR S489X homozygous (CF) mutant mice reportedly die of intestinal obstruction by 40 days of age, significantly limiting their usefulness as a model for the human disease. Because the period of highest mortality is during the week after weaning, we hypothesized that providing a low-residue liquid diet would improve survival and growth. When 99 CF mice that survived to 10 days of age were fed Peptamen (Clintec Nutrition), an elemental liquid diet, and housed on corn-cob bedding, 88% of them survived to maturity (50 days). The diet causes only minor histologic and ion transport changes in the intestines of normal mice and does not reduce growth rate or size. CF mice raised on Peptamen continue to display severe pathological changes in the intestine and completely lack a adenosine 3',5'-cyclic monophosphate-inducible chloride current in the cecum. This combination of dietary and bedding changes provides a reliable method for keeping CF mice alive well into adulthood and will be useful for the evaluation of the effect and duration of potential therapies for CF.

scholarly journals Near-infrared upconversion–activated CRISPR-Cas9 system: A remote-controlled gene editing platform

2019 ◽  
Vol 5 (4) ◽  
pp. eaav7199 ◽  
Author(s):  
Yongchun Pan ◽  
Jingjing Yang ◽  
Xiaowei Luan ◽  
Xinli Liu ◽  
Xueqing Li ◽  
...  
Keyword(s):  
Gene Editing ◽  
Near Infrared ◽  
Cancer Therapeutics ◽  
Guide Rna ◽  
System A ◽  
Nir Light ◽  
Targeted Gene Editing ◽  
Daunting Challenge

As an RNA-guided nuclease, CRISPR-Cas9 offers facile and promising solutions to mediate genome modification with respect to versatility and high precision. However, spatiotemporal manipulation of CRISPR-Cas9 delivery remains a daunting challenge for robust effectuation of gene editing both in vitro and in vivo. Here, we designed a near-infrared (NIR) light–responsive nanocarrier of CRISPR-Cas9 for cancer therapeutics based on upconversion nanoparticles (UCNPs). The UCNPs served as “nanotransducers” that can convert NIR light (980 nm) into local ultraviolet light for the cleavage of photosensitive molecules, thereby resulting in on-demand release of CRISPR-Cas9. In addition, by preparing a single guide RNA targeting a tumor gene (polo-like kinase-1), our strategies have successfully inhibited the proliferation of tumor cell via NIR light–activated gene editing both in vitro and in vivo. Overall, this exogenously controlled method presents enormous potential for targeted gene editing in deep tissues and treatment of a myriad of diseases.

scholarly journals Variation in zygotic CRISPR/Cas9 gene editing outcomes generates novel reporter and deletion alleles at the Gdf11 locus

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jill M. Goldstein ◽  
Austin Valido ◽  
Jordan P. Lewandowski ◽  
Ryan G. Walker ◽  
Melanie J. Mills ◽  
...  
Keyword(s):  
Gene Editing ◽  
Genetically Engineered ◽  
Lymphoid Cells ◽  
Null Alleles ◽  
Gfp Expression ◽  
Pcr Screening ◽  
C Terminus ◽  
Cas Gene

AbstractRecent advances in CRISPR/Cas gene editing technology have significantly expanded the possibilities and accelerated the pace of creating genetically engineered animal models. However, CRISPR/Cas-based strategies designed to precisely edit the genome can often yield unintended outcomes. Here, we report the use of zygotic CRISPR/Cas9 injections to generate a knock-in GFP reporter mouse at the Gdf11 locus. Phenotypic and genomic characterization of founder animals from these injections revealed a subset that contained the correct targeting event and exhibited GFP expression that, within the hematopoietic system, was restricted predominantly to lymphoid cells. Yet, in another subset of founder mice, we detected aberrant integration events at the target site that dramatically and inaccurately shifted hematopoietic GFP expression from the lymphoid to the myeloid lineage. Additionally, we recovered multiple Gdf11 deletion alleles that modified the C-terminus of the GDF11 protein. When bred to homozygosity, most of these alleles recapitulated skeletal phenotypes reported previously for Gdf11 knockout mice, suggesting that these represent null alleles. However, we also recovered one Gdf11 deletion allele that encodes a novel GDF11 variant protein (“GDF11-WE”) predicted to contain two additional amino acids (tryptophan (W) and glutamic acid (E)) at the C-terminus of the mature ligand. Unlike the other Gdf11 deletion alleles recovered in this study, homozygosity for the Gdf11WE allele did not phenocopy Gdf11 knockout skeletal phenotypes. Further investigation using in vivo and in vitro approaches demonstrated that GDF11-WE retains substantial physiological function, indicating that GDF11 can tolerate at least some modifications of its C-terminus and providing unexpected insights into its biochemical activities. Altogether, our study confirms that one-step zygotic injections of CRISPR/Cas gene editing complexes provide a quick and powerful tool to generate gene-modified mouse models. Moreover, our findings underscore the critical importance of thorough characterization and validation of any modified alleles generated by CRISPR, as unintended on-target effects that fail to be detected by simple PCR screening can produce substantially altered phenotypic readouts.

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