scholarly journals Inhibition of histone deacetylase 1 (HDAC1) and HDAC2 enhances CRISPR/Cas9 genome editing

2019 ◽  
Vol 48 (2) ◽  
pp. 517-532 ◽  
Author(s):  
Bin Liu ◽  
Siwei Chen ◽  
Anouk La Rose ◽  
Deng Chen ◽  
Fangyuan Cao ◽  
...  
Keyword(s):  
Gene Editing ◽  
Gene Knockout ◽  
Rapid Development ◽  
Open Chromatin ◽  
Chromatin Compaction ◽  
Histone Deacetylase 1 ◽  
Cas9 Protein ◽  
Target Dna ◽  
Histone Modifiers ◽  
Low Efficiency

Abstract Despite the rapid development of CRISPR/Cas9-mediated gene editing technology, the gene editing potential of CRISPR/Cas9 is hampered by low efficiency, especially for clinical applications. One of the major challenges is that chromatin compaction inevitably limits the Cas9 protein access to the target DNA. However, chromatin compaction is precisely regulated by histone acetylation and deacetylation. To overcome these challenges, we have comprehensively assessed the impacts of histone modifiers such as HDAC (1–9) inhibitors and HAT (p300/CBP, Tip60 and MOZ) inhibitors, on CRISPR/Cas9 mediated gene editing efficiency. Our findings demonstrate that attenuation of HDAC1, HDAC2 activity, but not other HDACs, enhances CRISPR/Cas9-mediated gene knockout frequencies by NHEJ as well as gene knock-in by HDR. Conversely, inhibition of HDAC3 decreases gene editing frequencies. Furthermore, our study showed that attenuation of HDAC1, HDAC2 activity leads to an open chromatin state, facilitates Cas9 access and binding to the targeted DNA and increases the gene editing frequencies. This approach can be applied to other nucleases, such as ZFN and TALEN.

scholarly journals Inhibition of Histone deacetylase 1 (HDAC1) and HDAC2 enhances CRISPR/Cas9 genome editing

2019 ◽  
Author(s):  
Bin Liu ◽  
Siwei Chen ◽  
Anouk La Rose ◽  
Deng Chen ◽  
Fangyuan Cao ◽  
...  
Keyword(s):  
Gene Editing ◽  
Gene Knockout ◽  
Rapid Development ◽  
Open Chromatin ◽  
Chromatin Compaction ◽  
Histone Deacetylase 1 ◽  
Cas9 Protein ◽  
Target Dna ◽  
Histone Modifiers ◽  
Low Efficiency

AbstractDespite the rapid development of CRISPR/Cas9-mediated gene editing technology, the gene editing potential of CRISPR/Cas9 is hampered by low efficiency, especially for clinical applications. One of the major challenges is that chromatin compaction inevitably limits the Cas9 protein access to the target DNA. However, chromatin compaction is precisely regulated by histone acetylation and deacetylation. To overcome these challenges, we have comprehensively assessed the impacts of histone modifiers such as HDAC (1-9) inhibitors and HAT (p300/CBP, Tip60 and MOZ) inhibitors, on CRISPR/Cas9 mediated gene editing efficiency. Our findings demonstrate that attenuation of HDAC1, HDAC2 activity, but not other HDACs, enhances CRISPR/Cas9-mediated gene knockout frequencies by NHEJ as well as gene knock-in by HDR. Conversely, inhibition of HDAC3 decreases gene editing frequencies. Furthermore, our study showed that attenuation of HDAC1, HDAC2 activity leads to an open chromatin state, facilitates Cas9 access and binding to the targeted DNA and increases the gene editing frequencies. This approach can be applied to other nucleases, such as ZFN and TALEN.

CRISPR/Cas9 gene editing: Applications in Parasitology

QJM ◽  
2020 ◽  
Vol 113 (Supplement_1) ◽  
Author(s):  
K E Khalifa
Keyword(s):  
Cancer Research ◽  
Gene Editing ◽  
Disease Models ◽  
Research Development ◽  
Gene Drive ◽  
Water Flea ◽  
Knock Out ◽  
Cas9 Protein ◽  
Target Dna ◽  
Made In

Abstract Clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 9 (CRISPR-Cas9) technology is adapted from the bacterial immune system and has rapidly revolutionized the editing of many genomes. This system is based on two components: an RNA-guided endonuclease, classically the Cas9 protein, and an RNA molecule that guides (gRNA) the nuclease to a complementary locus in the target DNA. The technique has allowed researchers to do gene knock-out, knock-in, insertions and deletions in cell lines and animals in rapid efficient way. The technique has many applications in the study of genes function, gene drive, cancer research, development of disease models and new therapeutic targets and crops enhancement. In the field of parasitology, using CRISPR/Cas9, significant advancements have been made in genome editing of Plasmodium spp, Leishmania, Typanosoma cruzi, and Toxoplasma gondii, as well as vectors like the mosquito, water flea and ticks. The aim of the talk is to present the basics of CRISPR/Cas9 and summarize some of its applications in the field of parasitology

scholarly journals Application of CRISPR/Cas9 technology in wild apple (Malus sieverii) for paired sites gene editing

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Yan Zhang ◽  
Ping Zhou ◽  
Tohir A. Bozorov ◽  
Daoyuan Zhang
Keyword(s):  
Abiotic Stress ◽  
Human Activities ◽  
Genetic Improvement ◽  
Gene Editing ◽  
New Technologies ◽  
Tianshan Mountains ◽  
Biotic And Abiotic Stress ◽  
Guide Rna ◽  
Cas9 Protein ◽  
Albino Phenotype

Abstract Background Xinjiang wild apple is an important tree of the Tianshan Mountains, and in recent years, it has undergone destruction by many biotic and abiotic stress and human activities. It is necessary to use new technologies to research its genomic function and molecular improvement. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability varies depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Results In this study, we used 2 systems of vectors with paired sgRNAs targeting to MsPDS. As expected, we successfully induced the albino phenotype of calli and buds in both systems. Conclusions We conclude that CRISPR/Cas9 is a powerful system for editing the wild apple genome and expands the range of plants available for gene editing.

scholarly journals Carboxylated branched poly(β-amino ester) nanoparticles enable robust cytosolic protein delivery and CRISPR-Cas9 gene editing

2019 ◽  
Vol 5 (12) ◽  
pp. eaay3255 ◽  
Author(s):  
Yuan Rui ◽  
David R. Wilson ◽  
John Choi ◽  
Mahita Varanasi ◽  
Katie Sanders ◽  
...  
Keyword(s):  
Protein Delivery ◽  
Gene Editing ◽  
Gene Knockout ◽  
Cell Types ◽  
Endosomal Escape ◽  
Biological Research ◽  
Amino Ester ◽  
Cytosolic Protein

Efficient cytosolic protein delivery is necessary to fully realize the potential of protein therapeutics. Current methods of protein delivery often suffer from low serum tolerance and limited in vivo efficacy. Here, we report the synthesis and validation of a previously unreported class of carboxylated branched poly(β-amino ester)s that can self-assemble into nanoparticles for efficient intracellular delivery of a variety of different proteins. In vitro, nanoparticles enabled rapid cellular uptake, efficient endosomal escape, and functional cytosolic protein release into cells in media containing 10% serum. Moreover, nanoparticles encapsulating CRISPR-Cas9 ribonucleoproteins (RNPs) induced robust levels of gene knock-in (4%) and gene knockout (>75%) in several cell types. A single intracranial administration of nanoparticles delivering a low RNP dose (3.5 pmol) induced robust gene editing in mice bearing engineered orthotopic murine glioma tumors. This self-assembled polymeric nanocarrier system enables a versatile protein delivery and gene editing platform for biological research and therapeutic applications.

Genome mutation after introduction of the gene editing by electroporation of Cas9 protein (GEEP) system in matured oocytes and putative zygotes

2019 ◽  
Vol 55 (4) ◽  
pp. 237-242 ◽  
Author(s):  
Maki Hirata ◽  
Fuminori Tanihara ◽  
Manita Wittayarat ◽  
Takayuki Hirano ◽  
Nhien Thi Nguyen ◽  
...  
Keyword(s):  
Gene Editing ◽  
Cas9 Protein

scholarly journals One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs

Cell Research ◽  
2017 ◽  
Vol 27 (7) ◽  
pp. 933-945 ◽  
Author(s):  
Erwei Zuo ◽  
Yi-Jun Cai ◽  
Kui Li ◽  
Yu Wei ◽  
Bang-An Wang ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Gene Editing ◽  
Gene Knockout ◽  
Gene Knockout Mice ◽  
One Step ◽  
Step Generation

scholarly journals Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells

2018 ◽  
Vol 215 (3) ◽  
pp. 985-997 ◽  
Author(s):  
Akiko Seki ◽  
Sascha Rutz
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Gene Editing ◽  
Target Gene ◽  
Gene Knockout ◽  
Great Promise ◽  
Next Generation ◽  
Primary Mouse

CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated protein 9) has become the tool of choice for generating gene knockouts across a variety of species. The ability for efficient gene editing in primary T cells not only represents a valuable research tool to study gene function but also holds great promise for T cell–based immunotherapies, such as next-generation chimeric antigen receptor (CAR) T cells. Previous attempts to apply CRIPSR/Cas9 for gene editing in primary T cells have resulted in highly variable knockout efficiency and required T cell receptor (TCR) stimulation, thus largely precluding the study of genes involved in T cell activation or differentiation. Here, we describe an optimized approach for Cas9/RNP transfection of primary mouse and human T cells without TCR stimulation that results in near complete loss of target gene expression at the population level, mitigating the need for selection. We believe that this method will greatly extend the feasibly of target gene discovery and validation in primary T cells and simplify the gene editing process for next-generation immunotherapies.

scholarly journals PDTM-26. NOVEL SHARED AND DISTINCT EPIGENOMIC MECHANISMS OF G34R AND K27M MUTATIONS IN CHILDHOOD GLIOMA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi192-vi193
Author(s):  
Michael Chen ◽  
Kelly Bush ◽  
Nichole Lewis ◽  
Vanessa Cervantes ◽  
Paul Knoepfler
Keyword(s):  
Gene Editing ◽  
Gene Knockout ◽  
Point Mutations ◽  
Glioma Cells ◽  
Driver Mutations ◽  
High Grade ◽  
Shared Key ◽  
Induced Changes ◽  
Open Question

Abstract Alterations in histone H3.3 are common driver mutations in high-grade pediatric gliomas, but the central oncogenic mechanisms remain an open question. To identify important mutant H3.3 effectors, we used CRISPR-Cas9 to precisely introduce H3.3 K27M and G34R mutations into previously H3.3-wildtype human astrocyte and glioma cells, while in parallel reverting mutations in glioma cells back to wildtype. K27M and G34R mutations invoked some strikingly similar epigenomic effects supporting a new model in which some major aspects of their oncogenic functions are shared. For instance, both K27M and G34R induced changes at many of the same genomic loci in specific histone marks, with the largest changes in H3K27me3 including in particular within super-enhancers, which also exhibited perturbed transcriptional function. K27M and G34R mutations induced some gene expression changes that were unique to each mutation, but both mutations changed similar functional ontological clusters and ASCL1 is a shared key putative effector. H3.3 mutant glioma cells are sensitive to ASCL1 knockdown or overexpression, resulting in cell viability that is reduced or increased, respectively. Comparison of our panel of glioma cells gene-edited with precise point mutations to edited glioma cells in other studies that performed gene knockout or overexpression reveals striking differences in the resulting phenotypes. We also determined that certain drugs exhibited specificity to H3.3 mutation-bearing cells including DAPT, JQ1, and ONC201. In vivo, we found that reversion of K27M to WT in glioma cells significantly reduced tumorigenicity in mouse xenograft assays and introduction of G34R mutations in previously WT glioma cells increased tumor growth. Overall, gene editing of gliomas and comparison of otherwise isogenic sets of cells defines both distinct and shared gliomagenesis mechanisms that can be targeted for development of oncohistone-based therapeutics.

scholarly journals A review of electromechanical actuators for More/All Electric aircraft systems

2017 ◽  
Vol 232 (22) ◽  
pp. 4128-4151 ◽  
Author(s):  
Guan Qiao ◽  
Geng Liu ◽  
Zhenghong Shi ◽  
Yawen Wang ◽  
Shangjun Ma ◽  
...  
Keyword(s):  
Rapid Development ◽  
Performance Testing ◽  
Future Research ◽  
Mechanical Transmission ◽  
Direct Drive ◽  
Electromechanical Actuators ◽  
Electromechanical Actuator ◽  
Aircraft Systems ◽  
Electric Aircraft ◽  
Low Efficiency

Conventional hydraulic actuators in aircraft systems are high maintenance and more vulnerable to high temperatures and pressures. This usually leads to high operating costs and low efficiency. With the rapid development of More/All Electric technology, power-by-wire actuators are being broadly employed to improve the maintainability, reliability, and manoeuvrability of future aircraft. This paper reviews the published application and development of the airborne linear electromechanical actuator. First, the general configuration, merits, and limitations of the gear-drive electromechanical actuator and the direct-drive electromechanical actuator are analysed. Second, the development state of the electromechanical actuator testing systems is elaborated in three aspects, namely the performance testing based on room temperature, testing in a thermal vacuum environment, and iron bird. Common problems and tendencies of the testing systems are summarized. Key technologies and research challenges are revealed in terms of fault-tolerant motor, high-thrust mechanical transmission, multidisciplinary modelling, thermal management, and thermal analysis. Finally, the trend for future electromechanical actuators in More/All Electric Aircraft applications is summarized, and future research on the airborne linear electromechanical actuators is discussed.

How to Recommend by Online Lifestyle Tagging (OLT)

2014 ◽  
Vol 13 (06) ◽  
pp. 1183-1209 ◽  
Author(s):  
Yu Pan ◽  
Lijuan Luo ◽  
Dan Liu ◽  
Li Gao ◽  
Xiaobo Xu ◽  
...  
Keyword(s):  
User Behavior ◽  
Rapid Development ◽  
Personalized Recommendation ◽  
Chinese Consumers ◽  
Online Consumer Behavior ◽  
Marketing Tool ◽  
Online Lifestyle ◽  
Low Efficiency ◽  
Using Data ◽  
Fierce Competition

With the rapid development of the Internet, the online shopping market expands constantly. Inspired by fierce competition and complex and diverse consumer demand, personalized recommendation has become an effective marketing tool for e-commerce enterprises. However, the existing recommendation methods based on online consumer behavior or preferences are characterized by poor accuracy and low efficiency. The paper mainly conducts three studies, the study1 proves that seven online lifestyles, which are "Comfortable, Entertainment, Luxury, Tradition & Conservation, Rational, Fashion Sense, and Social Activities", affect Chinese consumers' purchase. However, the different online lifestyles have different effects on purchase, thus the response rates of recommending. The study2 proposes a new personalized recommendation method "online lifestyle tagging (OLT)" based on online lifestyle and user behavior tags to identify online lifestyles. In the study3, the efficiency of OLT is tested and verified using data collected from enterprises, it suggests that OLT has a significantly higher response rate than traditional behavior-based methods. This study demonstrates that OLT improves the accuracy and efficiency of personalized recommendation, and thus contributes to the theory of personalized recommendation and marketing methods based on lifestyle.

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