scholarly journals Simple and large-scale chromosomal engineering of mouse zygotes via in vitro and in vivo electroporation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Satoru Iwata ◽  
Hitomi Nakadai ◽  
Daisuke Fukushi ◽  
Mami Jose ◽  
Miki Nagahara ◽  
...  
Keyword(s):  
Large Scale ◽  
Genome Engineering ◽  
Genetically Engineered ◽  
Guide Rna ◽  
Chromosomal Inversions ◽  
Genetically Engineered Mice ◽  
Cas9 Protein ◽  
Mouse Zygotes

Abstract The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has facilitated dramatic progress in the field of genome engineering. Whilst microinjection of the Cas9 protein and a single guide RNA (sgRNA) into mouse zygotes is a widespread method for producing genetically engineered mice, in vitro and in vivo electroporation (which are much more convenient strategies) have recently been developed. However, it remains unknown whether these electroporation methods are able to manipulate genomes at the chromosome level. In the present study, we used these techniques to introduce chromosomal inversions of several megabases (Mb) in length in mouse zygotes. Using in vitro electroporation, we successfully introduced a 7.67 Mb inversion, which is longer than any previously reported inversion produced using microinjection-based methods. Additionally, using in vivo electroporation, we also introduced a long chromosomal inversion by targeting an allele in F1 hybrid mice. To our knowledge, the present study is the first report of target-specific chromosomal inversions in mammalian zygotes using electroporation.

scholarly journals Large-scale GMP-compliant CRISPR-Cas9–mediated deletion of the glucocorticoid receptor in multivirus-specific T cells

2020 ◽  
Vol 4 (14) ◽  
pp. 3357-3367 ◽  
Author(s):  
Rafet Basar ◽  
May Daher ◽  
Nadima Uprety ◽  
Elif Gokdemir ◽  
Abdullah Alsuliman ◽  
...  
Keyword(s):  
T Cells ◽  
Glucocorticoid Receptor ◽  
Large Scale ◽  
Genetically Engineered ◽  
Cell Transplant ◽  
Hematopoietic Stem ◽  
Management Of Complications ◽  
Rapid Generation

Abstract Virus-specific T cells have proven highly effective for the treatment of severe and drug-refractory infections after hematopoietic stem cell transplant (HSCT). However, the efficacy of these cells is hindered by the use of glucocorticoids, often given to patients for the management of complications such as graft-versus-host disease. To address this limitation, we have developed a novel strategy for the rapid generation of good manufacturing practice (GMP)–grade glucocorticoid-resistant multivirus-specific T cells (VSTs) using clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) gene-editing technology. We have shown that deleting the nuclear receptor subfamily 3 group C member 1 (NR3C1; the gene encoding for the glucocorticoid receptor) renders VSTs resistant to the lymphocytotoxic effect of glucocorticoids. NR3C1-knockout (KO) VSTs kill their targets and proliferate successfully in the presence of high doses of dexamethasone both in vitro and in vivo. Moreover, we developed a protocol for the rapid generation of GMP-grade NR3C1 KO VSTs with high on-target activity and minimal off-target editing. These genetically engineered VSTs promise to be a novel approach for the treatment of patients with life-threatening viral infections post-HSCT on glucocorticoid therapy.

scholarly journals Generation and Characterization of Typhoid Toxin-Neutralizing Human Monoclonal Antibodies

2020 ◽  
Vol 88 (10) ◽  
Author(s):  
Xuyao Jiao ◽  
Sarah Smith ◽  
Gabrielle Stack ◽  
Qi Liang ◽  
Allan Bradley ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Typhoid Fever ◽  
Severe Disease ◽  
Genetically Engineered ◽  
Human Monoclonal Antibodies ◽  
Genetically Engineered Mice ◽  
Typhoid Toxin

ABSTRACT Typhoid toxin is a virulence factor of Salmonella enterica serovar Typhi, the causative agent of typhoid fever, and is thought to be responsible for the symptoms of severe disease. This toxin has a unique A2B5 architecture with two active subunits, the ADP ribosyl transferase PltA and the DNase CdtB, linked to a pentameric B subunit, which is alternatively made of PltB or PltC. Here, we describe the generation and characterization of typhoid toxin-neutralizing human monoclonal antibodies by immunizing genetically engineered mice that have a full set of human immunoglobulin variable region genes. We identified several monoclonal antibodies with strong in vitro and in vivo toxin-neutralizing activity and different mechanisms of toxin neutralization. These antibodies could serve as the basis for the development of novel therapeutic strategies against typhoid fever.

scholarly journals Discrete Role for Cytosolic Phospholipase A2α in Platelets

2002 ◽  
Vol 196 (3) ◽  
pp. 349-357 ◽  
Author(s):  
Dennis A. Wong ◽  
Yoshihiro Kita ◽  
Naonori Uozumi ◽  
Takao Shimizu
Keyword(s):  
Therapeutic Potential ◽  
Genetically Engineered ◽  
Collagen Receptors ◽  
Cytosolic Phospholipase ◽  
Genetically Engineered Mice ◽  
Cytosolic Pla2 ◽  
In Vivo Effects ◽  
Deficient Mice

Among several different types of phospholipase A2 (PLA2), cytosolic PLA2 (cPLA2)α and group IIA (IIA) secretory PLA2 (sPLA2) have been studied intensively. To determine the discrete roles of cPLA2α in platelets, we generated two sets of genetically engineered mice (cPLA2α−/−/sPLA2-IIA−/− and cPLA2α−/−/sPLA2-IIA+/+) and compared their platelet function with their respective wild-type C57BL/6J mice (cPLA2α+/+/sPLA2-IIA−/−) and C3H/HeN (cPLA2α+/+/sPLA2-IIA+/+). We found that cPLA2α is needed for the production of the vast majority of thromboxane (TX)A2 with collagen stimulation of platelets. In cPLA2α-deficient mice, however, platelet aggregation in vitro is only fractionally decreased because small amounts of TX produced by redundant phospholipase enzymes sufficiently preserve aggregation. In comparison, adenosine triphosphate activation of platelets appears wholly independent of cPLA2α and sPLA2-IIA for aggregation or the production of TX, indicating that these phospholipases are specifically linked to collagen receptors. However, the lack of high levels of TX limiting vasoconstriction explains the in vivo effects seen: increased bleeding times and protection from thromboembolism. Thus, cPLA2α plays a discrete role in the collagen-stimulated production of TX and its inhibition has a therapeutic potential against thromboembolism, with potentially limited bleeding expected.

scholarly journals Direct preparation of Cas9 ribonucleoprotein from E. coli for PCR-free seamless DNA assembly

2018 ◽  
Author(s):  
Wenqiang Li ◽  
Shuntang Li ◽  
Jie Qiao ◽  
Fei Wang ◽  
Yang Liu ◽  
...  
Keyword(s):  
Large Scale ◽  
Genome Engineering ◽  
Restriction Enzymes ◽  
General Strategy ◽  
Dna Assembly ◽  
E Coli ◽  
Assembly Method ◽  
Direct Preparation

AbstractCRISPR-Cas9 is a versatile and powerful genome engineering tool. Recently, Cas9 ribonucleoprotein (RNP) complexes have been used as promising biological tools with plenty of in vivo and in vitro applications, but there are by far no efficient methods to produce Cas9 RNP at large scale and low cost. Here, we describe a simple and effective approach for direct preparation of Cas9 RNP from E. coli by co-expressing Cas9 and target specific single guided RNAs. The purified RNP showed in vivo genome editing ability, as well as in vitro endonuclease activity that combines with an unexpected superior stability to enable routine uses in molecular cloning instead of restriction enzymes. We further develop a RNP-based PCR-free method termed Cas-Brick in a one-step or cyclic way for seamless assembly of multiple DNA fragments with high fidelity up to 99%. Altogether, our findings provide a general strategy to prepare Cas9 RNP and supply a convenient and cost-effective DNA assembly method as an invaluable addition to synthetic biological toolboxes.

scholarly journals Blocking gephyrin phosphorylation or microglia BDNF signaling prevents synapse loss and reduces infarct volume after ischemia

2020 ◽  
Author(s):  
Teresa Cramer ◽  
Raminder Gill ◽  
Zahra S Thirouin ◽  
Markus Vaas ◽  
Suchita Sampath ◽  
...  
Keyword(s):  
Hippocampal Formation ◽  
Infarct Volume ◽  
Point Mutations ◽  
Null Point ◽  
Genetically Engineered ◽  
Infarct Area ◽  
Synapse Plasticity ◽  
Genetically Engineered Mice

AbstractMicroglia interact with neurons to facilitate synapse plasticity; however, signal transducers between microglia and neuron remain unknown. Here, using in vitro organotypic hippocampal slice cultures and transient MCAO in genetically-engineered mice in vivo, we report that at 24 h post-ischemia microglia release BDNF to downregulate glutamatergic and GABAergic synapses within the peri-infarct area. Analysis of the CA1 hippocampal formation in vitro shows that proBDNF and mBDNF downregulate glutamatergic dendritic spines and gephyrin scaffold stability through p75NTR and TrkB receptors respectively. Post-MCAO, we report that in the peri- infarct area and in the corresponding contralateral hemisphere similar neuroplasticity occur through microglia activation and gephyrin phosphorylation at Ser268, Ser270 in vivo. Targeted deletion of the Bdnf gene in microglia or GphnS268A/S270A (phospho-null) point-mutations protect against ischemic brain damage, neuroinflamation and synapse downregulation normally seen post-MCAO. Collectively, we report that gephyrin phosphorylation and microglia derived BDNF faciliate synapse plasticity after transient ischemia.

scholarly journals Genome-wide CRISPR-Cas9 screen in E. coli identifies design rules for efficient targeting

2018 ◽  
Author(s):  
Belen Gutierrez ◽  
Jérôme Wong Ng ◽  
Lun Cui ◽  
Christophe Becavin ◽  
David Bikard
Keyword(s):  
Copy Number ◽  
Large Scale ◽  
Mixed Population ◽  
Target Sequence ◽  
Guide Rna ◽  
E Coli ◽  
Genome Wide ◽  
A Genome

AbstractThe main outcome of efficient CRISPR-Cas9 cleavage in the chromosome of bacteria is cell death. This can be conveniently used to eliminate specific genotypes from a mixed population of bacteria, which can be achieved both in vitro, e.g. to select mutants, or in vivo as an antimicrobial strategy. The efficiency with which Cas9 kills bacteria has been observed to be quite variable depending on the specific target sequence, but little is known about the sequence determinants and mechanisms involved. Here we performed a genome-wide screen of Cas9 cleavage in the chromosome of E. coli to determine the efficiency with which each guide RNA kills the cell. Surprisingly we observed a large-scale pattern where guides targeting some regions of the chromosome are more rapidly depleted than others. Unexpectedly, this pattern arises from the influence of degrading specific chromosomal regions on the copy number of the plasmid carrying the guide RNA library. After taking this effect into account, it is possible to train a neural network to predict Cas9 efficiency based on the target sequence. We show that our model learns different features than previous models trained on Eukaryotic CRISPR-Cas9 knockout libraries. Our results highlight the need for specific models to design efficient CRISPR-Cas9 tools in bacteria.

scholarly journals Prediction of activity of CRISPR-Cpf1 guide RNA via in vivo high-throughput screening

2021 ◽  
Author(s):  
gancheng wang ◽  
dan zhu ◽  
juan li ◽  
junyi wang ◽  
jianzhong xi
Keyword(s):  
Dna Cleavage ◽  
High Throughput Screening ◽  
Large Scale ◽  
Genome Engineering ◽  
Limited Information ◽  
Editing Efficiency ◽  
Guide Rna ◽  
Guide Rnas ◽  
Promising Tool

Background: CRISPR-cpf1 is a single RNA-guided endonuclease system, becoming a promising tool in both prokaryotic and eukaryotic genome engineering. The editing efficiency of Cpf1 based engineering still requires improvements. However, limited information regarding the relationship between guide RNA sequence and on-target activity is available. To address these challenges, we developed a screening platform based on the association of Acidaminococcus sp. Cpf1(AsCpf1) DNA cleavage with cellular lethality. Major results: In total, we measured the activities of 12,544 guide RNAs, and observed a substantial variation of the editing efficiency depending on the design of the sequence. Based on this large-scale dataset, we designed and implemented a comprehensive computational model to predict activities of guide RNAs. Through comparison using simulated and experimental data, our approach outperformed existing algorithms, enabling selection of efficient guide RNAs. Conclusions: We refine on-target design rules and isolate the important sequence features that contribute to DNA cleavage, that is, AH dimers at position1-8 of protospacer promoting Cas12a activity while TK, GB dimer playing an inhibitory role. We validate guide RNA affinities designed by our optimized rules in both E.coli and 293T cells.

scholarly journals Screening for functional circular RNAs using the CRISPR-Cas13 system

2020 ◽  
Author(s):  
Siqi Li ◽  
Xiang Li ◽  
Wei Xue ◽  
Lin Zhang ◽  
Shi-Meng Cao ◽  
...  
Keyword(s):  
Large Scale ◽  
Genome Engineering ◽  
Functional Study ◽  
Circular Rnas ◽  
Rna Targeting ◽  
Scale Levels ◽  
A Cell ◽  
Cell Type Specific

SummaryCircular RNAs (circRNAs) produced from back-spliced exons are widely expressed, but individual circRNA functions remain poorly understood due to inadequate methods, such as RNAi and genome engineering, in distinguishing overlapped exons in circRNAs from those in linear cognate mRNAs1,2. Here we report that the programable RNA-guided, RNA-targeting CRISPR-Cas13, RfxCas13d, effectively and specifically discriminates circRNAs from mRNAs, using guide (g)RNAs targeting sequences spanning the back-splicing junction (BSJ) sites featured in RNA circles. Using a lentiviral library that targets sequences across BSJ sites of highly expressed human circRNAs, we show that a group of circRNAs are important for cell growth mostly in a cell-type specific manner and that a common oncogenic circRNA, circFAM120A, promotes cell proliferation in vitro and in vivo by preventing FAM120A mRNA from binding the translation inhibitor IGF2BP2 for efficient translation. Application of RfxCas13d/BSJ-gRNA screening has also uncovered circMan1a2 with regulatory potential in mouse embryo preimplantation development. Together, these results establish CRISPR-RfxCas13d as a useful tool for the discovery and functional study of circRNAs at both individual and large-scale levels.

scholarly journals Precision modeling of gall bladder cancer patients in mice based on orthotopic implantation of organoid-derived tumor buds

Oncogenesis ◽  
2021 ◽  
Vol 10 (4) ◽  
Author(s):  
Shingo Kato ◽  
Kentaro Fushimi ◽  
Yuichiro Yabuki ◽  
Yoshiaki Maru ◽  
Sho Hasegawa ◽  
...  
Keyword(s):  
Gall Bladder ◽  
Ex Vivo ◽  
Tumor Development ◽  
Genetically Engineered ◽  
Intact Cells ◽  
Genetically Engineered Mice ◽  
Carcinogenesis Models ◽  
Subcutaneous Inoculation

AbstractGenetically engineered mice (GEM) are the gold standard for cancer modeling. However, strict recapitulation of stepwise carcinogenesis from a single tumor-initiating epithelial cell among genetically intact cells in adults is not feasible with the currently available techniques using GEM. In previous studies, we partially overcame this challenge by physically isolating organs from adult animals, followed by genetic engineering in organoids and subcutaneous inoculation in nude mice. Despite the establishment of suitable ex vivo carcinogenesis models for diverse tissues, tumor development remained ectopic and occurred under immunodeficient conditions. Further refinement was, therefore, necessary to establish ideal models. Given the poor prognosis and few models owing to the lack of gall bladder (GB)-specific Cre strain, we assumed that the development of authentic models would considerably benefit GB cancer research. Here, we established a novel model using GB organoids with mutant Kras and Trp53 loss generated in vitro by lentiviral Cre transduction and CRISPR/Cas9 gene editing, respectively. Organoid-derived subcutaneous tumor fragments were sutured to the outer surface of the GB in syngeneic mice, which developed orthotopic tumors that resembled human GB cancer in histological and transcriptional features. This model revealed the infiltration of similar subsets of immune cells in both subcutaneous and orthotopic tumors, confirming the appropriate immune environment during carcinogenesis. In addition, we accurately validated the in vivo efficacy of gemcitabine, a common therapeutic agent for GB cancer, in large cohorts. Taken together, this model may serve as a promising avatar of patients with GB cancer in drug discovery and precision medicine.

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