scholarly journals Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Masakazu Hashimoto ◽  
Tatsuya Takemoto
Keyword(s):  
Genetic Analysis ◽  
Genome Editing ◽  
High Throughput ◽  
Large Scale ◽  
Mouse Embryos ◽  
Mutant Mice ◽  
Highly Efficient ◽  
Mouse Zygotes ◽  
Time Required

Abstract Recent use of the CRISPR/Cas9 system has dramatically reduced the time required to produce mutant mice, but the involvement of a time-consuming microinjection step still hampers its application for high-throughput genetic analysis. Here we developed a simple, highly efficient and large-scale genome editing method, in which the RNAs for the CRISPR/Cas9 system are electroporated into zygotes rather than microinjected. We used this method to perform single-stranded oligodeoxynucleotide (ssODN)-mediated knock-in in mouse embryos. This method facilitates large-scale genetic analysis in the mouse.

scholarly journals A High-Throughput Gene-Silencing System for the Functional Assessment of Defense-Related Genes in Barley Epidermal Cells

2005 ◽  
Vol 18 (8) ◽  
pp. 755-761 ◽  
Author(s):  
D. Douchkov ◽  
D. Nowara ◽  
U. Zierold ◽  
P. Schweizer
Keyword(s):  
Gene Silencing ◽  
High Throughput ◽  
Gene Function ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Epidermal Cells ◽  
Powdery Mildew Fungus ◽  
Nonspecific Resistance ◽  
Knock Down ◽  
Highly Efficient

Large-scale gene silencing by RNA interference (RNAi) offers the possibility to address gene function in eukaryotic organisms at a depth unprecedented until recently. Although genome-wide RNAi approaches are being carried out in organisms like Caenorhabditis elegans, Drosophila spp. or human after the corresponding tools had been developed, knock-down of only single or a few genes by RNAi has been reported in plants thus far. Here, we present a method for high-throughput, transient-induced gene silencing (TIGS) by RNAi in barley epidermal cells that is based on biolistic transgene delivery. This method will be useful to address gene function of shoot epidermis resulting in cell-autonomous phenotypes such as resistance or susceptibility to the powdery-mildew fungus Blumeria graminis f. sp. hordei. Gene function in epidermal cell elongation, stomata regulation, or UV resistance might be addressed as well. Libraries of RNAi constructs can be built up by a new, cost-efficient method that combines highly efficient ligation and recombination by the Gateway cloning system. This method allows cloning of any blunt-ended DNA fragment without the need of adaptor sequences. The final RNAi destination vector was found to direct highly efficient RNAi, as reflected by complete knock-down of a cotransformed green fluorescent protein reporter gene as well as by complete phenolcopy of the recessive loss-of-function mlo resistance gene. By using this method, a role of the t-SNARE proteinin three types of durable, race-nonspecific resistance was observed.

scholarly journals Prime editing primarily induces undesired outcomes in mice

2020 ◽  
Author(s):  
Tomomi Aida ◽  
Jonathan J. Wilde ◽  
Lixin Yang ◽  
Yuanyuan Hou ◽  
Mengqi Li ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Frequency ◽  
Mouse Embryos ◽  
Biomedical Science ◽  
New Approach ◽  
Chemically Modified ◽  
Highly Efficient ◽  
Large Deletions

SummaryGenome editing has transformed biomedical science, but is still unpredictable and often induces undesired outcomes. Prime editing (PE) is a promising new approach due to its proposed flexibility and ability to avoid unwanted indels. Here, we show highly efficient PE-mediated genome editing in mammalian zygotes. Utilizing chemically modified guideRNAs, PE efficiently introduced 10 targeted modifications including substitutions, deletions, and insertions across 6 genes in mouse embryos. However, we unexpectedly observed a high frequency of undesired outcomes such as large deletions and found that these occurred more often than pure intended edits across all of the edits/genes. We show that undesired outcomes result from the double-nicking PE3 strategy, but that omission of the second nick largely ablates PE function. However, sequential double-nicking with PE3b, which is only applicable to a fraction of edits, eliminated undesired outcomes. Overall, our findings demonstrate the promising potential of PE for predictable, flexible, and highly efficient in vivo genome editing, but highlight the need for improved variations of PE before it is ready for widespread use.

scholarly journals A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids

2017 ◽  
Vol 4 (5) ◽  
pp. 170095 ◽  
Author(s):  
Tom Beneke ◽  
Ross Madden ◽  
Laura Makin ◽  
Jessica Valli ◽  
Jack Sunter ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Throughput ◽  
Large Scale ◽  
Leishmania Major ◽  
Fluorescent Protein ◽  
Genetic Manipulation ◽  
Gene Knockout ◽  
Guide Rna ◽  
Functional Studies

Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-associated gene 9 (Cas9) genome editing is set to revolutionize genetic manipulation of pathogens, including kinetoplastids. CRISPR technology provides the opportunity to develop scalable methods for high-throughput production of mutant phenotypes. Here, we report development of a CRISPR-Cas9 toolkit that allows rapid tagging and gene knockout in diverse kinetoplastid species without requiring the user to perform any DNA cloning. We developed a new protocol for single-guide RNA (sgRNA) delivery using PCR-generated DNA templates which are transcribed in vivo by T7 RNA polymerase and an online resource (LeishGEdit.net) for automated primer design. We produced a set of plasmids that allows easy and scalable generation of DNA constructs for transfections in just a few hours. We show how these tools allow knock-in of fluorescent protein tags, modified biotin ligase BirA*, luciferase, HaloTag and small epitope tags, which can be fused to proteins at the N- or C-terminus, for functional studies of proteins and localization screening. These tools enabled generation of null mutants in a single round of transfection in promastigote form Leishmania major , Leishmania mexicana and bloodstream form Trypanosoma brucei ; deleted genes were undetectable in non-clonal populations, enabling for the first time rapid and large-scale knockout screens.

scholarly journals MultiFRAGing: Rapid and Simultaneous Genotyping of Multiple Alleles in a Single Reaction

2019 ◽  
Author(s):  
Cassidy Petree ◽  
Gaurav K Varshney
Keyword(s):  
Genome Editing ◽  
High Throughput ◽  
Large Scale ◽  
Cost Effective ◽  
Fold Increase ◽  
Targeted Mutagenesis ◽  
Model Organisms ◽  
Multiple Alleles ◽  
Cost Effective Method ◽  
Single Reaction

AbstractThe powerful and simple RNA-guided CRISPR/Cas9 technology is a versatile genome editing tool that has revolutionized targeted mutagenesis. CRISPR-based genome editing has enabled large-scale functional genetic studies through the generation of gene knockouts in a variety of model organisms including zebrafish. CRISPR/Cas9 can also be used to target multiple genes simultaneously. One of the challenges associated with applying this technique to zebrafish in a high-throughput manner is the absence of a cost-effective method by which to identify mutants. To address this, we optimized the high-throughput, high-resolution fluorescent PCR-based fragment analysis method to develop MultiFRAGing, a robust and cost-effective method for genotyping of multiple targets in a single reaction. Our approach can identify indels in 4 targets from a single reaction, which represents a four-fold increase in genotyping throughput. This method can be used by any laboratory with access to capillary electrophoresis based sequencing equipment.

scholarly journals Efficient and Practical Sample Pooling for High-Throughput PCR Diagnosis of COVID-19

2020 ◽  
Author(s):  
Haran Shani-Narkiss ◽  
Omri David Gilday ◽  
Nadav Yayon ◽  
Itamar Daniel Landau
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Diagnostic Testing ◽  
Target Frequency ◽  
Public Health Agencies ◽  
Highly Efficient ◽  
Pcr Diagnosis ◽  
Sample Pooling ◽  
Pooling Strategies ◽  
Technical Advances

AbstractIn the global effort to combat the COVID-19 pandemic, governments and public health agencies are striving to rapidly increase the volume and rate of diagnostic testing. The most common form of testing today employs Polymerase Chain Reaction in order to identify the presence of viral RNA in individual patient samples one by one. This process has become one of the most significant bottlenecks to increased testing, especially due to reported shortages in the chemical reagents needed in the PCR reaction.Recent technical advances have enabled High-Throughput PCR, in which multiple samples are pooled into one tube. Such methods can be highly efficient, saving large amounts of time and reagents. However, their efficiency is highly dependent on the frequency of positive samples, which varies significantly across regions and even within regions as testing criterion and conditions change.Here, we present two possible optimized pooling strategies for diagnostic SARS-CoV-2 testing on large scales, both addressing dynamic conditions. In the first, we employ a simple information-theoretic heuristic to derive a highly efficient re-pooling protocol: an estimate of the target frequency determines the initial pool size, and any subsequent pools found positive are re-pooled at half-size and tested again. In the range of very rare target (<0.05), this approach can reduce the number of necessary tests dramatically, for example, achieving a reduction by a factor of 50 for a target frequency of 0.001. The second method is a simpler approach of optimized one-time pooling followed by individual tests on positive pools. We show that this approach is just as efficient for moderate target-product frequencies (0.05<0.2), for example, achieving a two-fold in the number of when the frequency of positive samples is 0.07.These strategies require little investment, and they offer a significant reduction in the amount of materials, equipment and time needed to test large numbers of samples. We show that both these pooling strategies are roughly comparable to the absolute upper-bound efficiency given by Shannon’s source coding theorem. We compare our strategies to the naïve way of testing and to alternative matrix-pooling methods. Most importantly, we offer straightforward, practical pooling instructions for laboratories that perform large scale PCR assays to diagnose SARS-CoV-2 viral particles. These two pooling strategies may offer ways to alleviate the bottleneck currently preventing massive expansion of SARS-CoV-2 testing around the world.

scholarly journals gEL DNA, a cloning- and PCR-free method for CRISPR-based multiplexed genome editing

2020 ◽  
Author(s):  
Paola Randazzo ◽  
Jean-Marc Daran ◽  
Pascale Daran-Lapujade
Keyword(s):  
Genome Editing ◽  
Large Scale ◽  
Yeast Species ◽  
T7 Rna Polymerase ◽  
Dna Fragments ◽  
Linear Dna ◽  
Highly Efficient ◽  
Yeast Strains ◽  
Dna Editing ◽  
Expression Plasmids

AbstractEven for the genetically accessible yeast Saccharomyces cerevisiae, the CRISPR/Cas DNA editing technology has strongly accelerated and facilitated strain construction. Several methods have been validated for fast and highly efficient single editing events and diverse approaches for multiplex genome editing have been described in literature by means of Cas9 or Cas12a endonucleases and their associated gRNAs. The gRNAs used to guide the Cas endonuclease to the editing site are typically expressed from plasmids using native PolII or PolIII RNA polymerases. These gRNA-expression plasmids require laborious, time-consuming cloning steps, which hampers their implementation for academic and applied purposes. In this study, we explore the potential of expressing gRNA from linear DNA fragments using the T7 RNA polymerase (T7RNAP) for single and multiplex genome editing in S. cerevisiae. Using Cas12a, this work demonstrates that transforming short, linear DNA fragments encoding gRNAs in yeast strains expressing T7RNAP promotes highly efficient single DNA editing. These DNA fragments can be custom-ordered, which makes this approach highly suitable for high-throughput strain construction. This work expands the CRISPR-toolbox for large-scale strain construction programs in S. cerevisiae and promises to be relevant for other, less genetically accessible yeast species.

scholarly journals Highly efficient and safe genome editing by CRISPR-Cas12a using CRISPR RNA with a ribosyl-2′-O-methylated uridinylate-rich 3′-overhang in mouse zygotes

2020 ◽  
Vol 52 (11) ◽  
pp. 1823-1830
Author(s):  
Dae-In Ha ◽  
Jeong Mi Lee ◽  
Nan-Ee Lee ◽  
Daesik Kim ◽  
Jeong-Heon Ko ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Animal Models ◽  
Genome Editing ◽  
Effector Proteins ◽  
Eukaryotic Cells ◽  
Aav Vector ◽  
Chemically Modified ◽  
Highly Efficient ◽  
Mouse Zygotes ◽  
Vector Delivery

AbstractThe CRISPR-Cas12a system has been developed to harness highly specific genome editing in eukaryotic cells. Given the relatively small sizes of Cas12a genes, the system has been suggested to be most applicable to gene therapy using AAV vector delivery. Previously, we reported that a U-rich crRNA enabled highly efficient genome editing by the CRISPR-Cas12a system in eukaryotic cells. In this study, we introduced methoxyl modifications at C2 in riboses in the U-rich 3′-overhang of crRNA. When mixed with Cas12a effector proteins, the ribosyl-2′-O-methylated (2-OM) U-rich crRNA enabled improvement of dsDNA digestibility. Moreover, the chemically modified U-rich crRNA achieved very safe and highly specific genome editing in murine zygotes. The engineered CRISPR-Cas12a system is expected to facilitate the generation of various animal models. Moreover, the engineered crRNA was evaluated to further improve a CRISPR genome editing toolset.

Accelerated Discovery of High-Refractive-Index Polyimides via First-Principles Molecular Modeling, Virtual High-Throughput Screening, and Data Mining

2019 ◽  
Author(s):  
Mohammad Atif Faiz Afzal ◽  
Mojtaba Haghighatlari ◽  
Sai Prasad Ganesh ◽  
Chong Cheng ◽  
Johannes Hachmann
Keyword(s):  
Data Mining ◽  
Refractive Index ◽  
High Throughput ◽  
First Principles ◽  
High Throughput Screening ◽  
Large Scale ◽  
Computational Study ◽  
High Refractive Index ◽  
Structural Features ◽  
Learning Program

<div>We present a high-throughput computational study to identify novel polyimides (PIs) with exceptional refractive index (RI) values for use as optic or optoelectronic materials. Our study utilizes an RI prediction protocol based on a combination of first-principles and data modeling developed in previous work, which we employ on a large-scale PI candidate library generated with the ChemLG code. We deploy the virtual screening software ChemHTPS to automate the assessment of this extensive pool of PI structures in order to determine the performance potential of each candidate. This rapid and efficient approach yields a number of highly promising leads compounds. Using the data mining and machine learning program package ChemML, we analyze the top candidates with respect to prevalent structural features and feature combinations that distinguish them from less promising ones. In particular, we explore the utility of various strategies that introduce highly polarizable moieties into the PI backbone to increase its RI yield. The derived insights provide a foundation for rational and targeted design that goes beyond traditional trial-and-error searches.</div>

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