Heritable gene editing using FT mobile guide RNAs and DNA viruses

Abstract Background The virus-induced genome editing (VIGE) system can be used to quickly identify gene functions and generate knock-out libraries as an alternative to the virus-induced gene silencing (VIGS). Although plant virus-mediated VIGE has been shown to have great application prospects, edited genes cannot be transferred to the next generations using this system, as viruses cannot enter into shoot apical meristem (SAM) in plants. Results We developed a novel cotton leaf crumple virus (CLCrV)-mediated VIGE system designed to target BRI1, GL2, PDS genes, and GUS transgene in A. thaliana by transforming Cas9 overexpression (Cas9-OE) A. thaliana. Given the deficiency of the VIGE system, ProYao::Cas9 and Pro35S::Cas9 A. thaliana were transformed by fusing 102 bp FT mRNAs with sgRNAs so as to explore the function of Flowering Locus T (FT) gene in delivering sgRNAs into SAM, thus avoiding tissue culture and stably acquiring heritable mutant offspring. Our results showed that sgRNAs fused with FT mRNA at the 5′ end (FT strategy) effectively enabled gene editing in infected plants and allowed the acquisition of mutations heritable by the next generation, with an efficiency of 4.35–8.79%. In addition, gene-edited offspring by FT-sgRNAs did not contain any components of the CLCrV genome. Conclusions FT strategy can be used to acquire heritable mutant offspring avoiding tissue culture and stable transformation based on the CLCrV-mediated VIGE system in A. thaliana.

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Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system

Plant Methods ◽

10.1186/1746-4811-8-27 ◽

2012 ◽

Vol 8 (1) ◽

pp. 27 ◽

Cited By ~ 15

Author(s):

John Tuttle ◽

Candace H Haigler ◽

Dominique Robertson

Keyword(s):

Gene Silencing ◽

Low Cost ◽

Cotton Leaf ◽

Virus Induced Gene Silencing ◽

Cotton Leaf Crumple Virus

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One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis

International Journal of Molecular Sciences ◽

10.3390/ijms22052249 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2249

Author(s):

Fuminori Tanihara ◽

Maki Hirata ◽

Nhien Thi Nguyen ◽

Osamu Sawamoto ◽

Takeshi Kikuchi ◽

...

Keyword(s):

Gene Editing ◽

Multiple Gene ◽

Knock Out ◽

Editing Efficiency ◽

Guide Rnas ◽

Cas9 Protein ◽

One Step ◽

Step Generation ◽

The One

Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting GGTA1, CMAH, and B4GALNT2 into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting GGTA1 and CMAH with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when GGTA1, CMAH, and B4GALNT2 were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.

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CRISPR as a Potential Technique to Reduce Suicide Risk

Journal of Student Research ◽

10.47611/jsrhs.v10i3.2240 ◽

2021 ◽

Vol 10 (3) ◽

Author(s):

Hidemi Zamora ◽

Javier Cornejo

Keyword(s):

Cystic Fibrosis ◽

Suicide Risk ◽

Gene Editing ◽

Design Tool ◽

High Importance ◽

Knock Out ◽

Guide Rna ◽

Guide Rnas ◽

The Past ◽

Rna Design

As suicide is the nineteenth leading cause of death worldwide, it is important to focus on discovering ways to reduce the risk of suicide-related death as much as possible. With CRISPR starting to become increasingly popular over the past few years, this gene editing technique has been used to study how to edit, turn off, or knock out multiple parts of the genome. However, research on genes related to diseases as cystic fibrosis or Alzheimer’s disease has been mainly prioritized and, even though they are of high importance as well, important issues such as suicide have been left into oblivion. Four genes have been proven to be key in influencing suicide risk, showing that not only environmental factors account for an increased possibility of death by this cause. Therefore, gene editing techniques such as CRISPR could be applied in order to knock out those genes and reduce this risk. This research used Synthego’s guide RNA design tool to predict how the use of CRISPR can be helpful in knocking out those four suicide-related genes and, consequently, in preventing suicide. The top-ranked guide RNAs for each gene were used, showing the best results possible and with the least number of off-targets, which, in turn, demonstrates the effectiveness of CRISPR as a potential technique to reduce the number of suicide-related deaths worldwide.

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Latest Advances of Virology Research Using CRISPR/Cas9-Based Gene-Editing Technology and Its Application to Vaccine Development

Viruses ◽

10.3390/v13050779 ◽

2021 ◽

Vol 13 (5) ◽

pp. 779

Author(s):

Man Teng ◽

Yongxiu Yao ◽

Venugopal Nair ◽

Jun Luo

Keyword(s):

Biological Sciences ◽

Gene Therapy ◽

Genetic Engineering ◽

Gene Function ◽

Viral Genome ◽

Gene Editing ◽

Vaccine Development ◽

Future Prospects ◽

Dna Viruses ◽

Viral Genomes

In recent years, the CRISPR/Cas9-based gene-editing techniques have been well developed and applied widely in several aspects of research in the biological sciences, in many species, including humans, animals, plants, and even in viruses. Modification of the viral genome is crucial for revealing gene function, virus pathogenesis, gene therapy, genetic engineering, and vaccine development. Herein, we have provided a brief review of the different technologies for the modification of the viral genomes. Particularly, we have focused on the recently developed CRISPR/Cas9-based gene-editing system, detailing its origin, functional principles, and touching on its latest achievements in virology research and applications in vaccine development, especially in large DNA viruses of humans and animals. Future prospects of CRISPR/Cas9-based gene-editing technology in virology research, including the potential shortcomings, are also discussed.

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Functional examination of lncRNAs in allotetraploid Gossypium hirsutum

BMC Genomics ◽

10.1186/s12864-021-07771-3 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Luyao Wang ◽

Jin Han ◽

Kening Lu ◽

Menglin Li ◽

Mengtao Gao ◽

...

Keyword(s):

Abiotic Stresses ◽

Expression Patterns ◽

Leaf Tissue ◽

Evolutionary Model ◽

Cotton Leaf ◽

Functional Screening ◽

Virus Induced Gene Silencing ◽

Individual Gene ◽

Cotton Species ◽

Allotetraploid Cotton

Abstract Background An evolutionary model using diploid and allotetraploid cotton species identified 80 % of non-coding transcripts in allotetraploid cotton as being uniquely activated in comparison with its diploid ancestors. The function of the lncRNAs activated in allotetraploid cotton remain largely unknown. Results We employed transcriptome analysis to examine the relationship between the lncRNAs and mRNAs of protein coding genes (PCGs) in cotton leaf tissue under abiotic stresses. LncRNA expression was preferentially associated with that of the flanking PCGs. Selected highly-expressed lncRNA candidates (n = 111) were subjected to a functional screening pilot test in which virus-induced gene silencing was integrated with abiotic stress treatment. From this low-throughput screen, we obtained candidate lncRNAs relating to plant height and tolerance to drought and other abiotic stresses. Conclusions Low-throughput screen is an effective method to find functional lncRNA for further study. LncRNAs were more active in abiotic stresses than PCG expression, especially temperature stress. LncRNA XLOC107738 may take a cis-regulatory role in response to environmental stimuli. The degree to which lncRNAs are constitutively expressed may impact expression patterns and functions on the individual gene level rather than in genome-wide aggregate.

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CRISPR–Cas9 gene editing as a tool for developing immunotherapy for cancer

10.31219/osf.io/dvr4t ◽

2021 ◽

Author(s):

Moataz Dowaidar

Keyword(s):

T Cells ◽

Immune Cells ◽

Peripheral Blood ◽

Gene Editing ◽

Genetically Engineered ◽

Cancer Resistance ◽

Knock Out ◽

Tumor Tissues ◽

Engineered T Cells ◽

Long Time

T cells following genome editing and transformation might be detectable in peripheral blood and tumor tissues for a long time, even more than a year. The types and diversity of T-cells in peripheral blood and tumor tissues changed following transfusion of genetically modified T-cells, and some highly suspected T-cells targeting cancer cells grew, increasing the proportion of such cells. Moreover, after getting genetically engineered T cells, anticancer cytokine secretion increased. T cells changed by gene editing have certain functions, at least from an immunological standpoint. The first clinical research using the CRISPR–Cas9 gene editing method for cancer resistance is more complicated: Using CRISPR–Cas9 gene editing technology to concurrently knock out, amplify, activate and reinfuse three genes in human immune cells. This therapeutic strategy is more demanding, because the changed immune cells have a wider target scope. The data suggest that the efficacy of gene editing in immune cells was 15–45%, and the modified cells could survive long in the peripheral blood and tumor tissues of patients. After three or four months, some T-cells became central T-cells. These encouraging findings pave the way for future experimental cancer research utilizing CRISPR technology.

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Rice Functional Genomics by Transposon Mutagenesis

Asia-Pacific Biotech News ◽

10.1142/s0219030302001933 ◽

2002 ◽

Vol 06 (24) ◽

pp. 930-935 ◽

Cited By ~ 8

Author(s):

Chang-deok Han

Keyword(s):

Tissue Culture ◽

Transposable Elements ◽

Functional Genomics ◽

Large Scale ◽

Expression Patterns ◽

Transposon Mutagenesis ◽

Transposon Tagging ◽

Genomic Sequences ◽

Knock Out ◽

Genetic Cross

Transposable elements are powerful mutagens. Along with genomic sequences, knock-out phenotypes and expression patterns are important information to elucidate the function of genes. In this review, I propose a strategy to develop tranposant lines on a large scale by combining genetic cross and tissue culture of Ac and Ds lines. Based on the facts that Ds tends to be inactive in F2 or later generation and Ds becomes reactivated via tissue culture, a large scale of transposants can be produced by tissue culture of seeds carrying Ac and inactive Ds. In this review, I describe limitations and considerations in operating transposon tagging systems in rice. Also, I discuss the efficiency of our gene trap system and technical procedures to clone Ds flanking DNA.

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Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1712963115 ◽

2018 ◽

Vol 115 (19) ◽

pp. 4903-4908 ◽

Cited By ~ 88

Author(s):

Hong-Xia Wang ◽

Ziyuan Song ◽

Yeh-Hsing Lao ◽

Xin Xu ◽

Jing Gong ◽

...

Keyword(s):

Gene Editing ◽

Transfection Efficiency ◽

Gene Activation ◽

Cell Types ◽

Biological Research ◽

Knock Out ◽

Safe Delivery ◽

Pegylated Nanoparticles

Effective and safe delivery of the CRISPR/Cas9 gene-editing elements remains a challenge. Here we report the development of PEGylated nanoparticles (named P-HNPs) based on the cationic α-helical polypeptide poly(γ-4-((2-(piperidin-1-yl)ethyl)aminomethyl)benzyl-l-glutamate) for the delivery of Cas9 expression plasmid and sgRNA to various cell types and gene-editing scenarios. The cell-penetrating α-helical polypeptide enhanced cellular uptake and promoted escape of pCas9 and/or sgRNA from the endosome and transport into the nucleus. The colloidally stable P-HNPs achieved a Cas9 transfection efficiency up to 60% and sgRNA uptake efficiency of 67.4%, representing an improvement over existing polycation-based gene delivery systems. After performing single or multiplex gene editing with an efficiency up to 47.3% in vitro, we demonstrated that P-HNPs delivering Cas9 plasmid/sgRNA targeting the polo-like kinase 1 (Plk1) gene achieved 35% gene deletion in HeLa tumor tissue to reduce the Plk1 protein level by 66.7%, thereby suppressing the tumor growth by >71% and prolonging the animal survival rate to 60% within 60 days. Capable of delivering Cas9 plasmids to various cell types to achieve multiplex gene knock-out, gene knock-in, and gene activation in vitro and in vivo, the P-HNP system offers a versatile gene-editing platform for biological research and therapeutic applications.

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The Diversity of Genetic Outcomes from CRISPR/Cas Gene Editing is Regulated by the Length of the Symmetrical Donor DNA Template

Genes ◽

10.3390/genes11101160 ◽

2020 ◽

Vol 11 (10) ◽

pp. 1160

Author(s):

Amanda M. Hewes ◽

Brett M. Sansbury ◽

Eric B. Kmiec

Keyword(s):

Gene Editing ◽

Bacterial Cells ◽

Knock Out ◽

Viral Genomes ◽

Homology Directed Repair ◽

Established Cell ◽

Dna Template ◽

Donor Template ◽

Eukaryotic Genomes ◽

Cas Gene

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas gene editing systems have enabled molecular geneticists to manipulate prokaryotic and eukaryotic genomes with greater efficiency and precision. CRISPR/Cas provides adaptive immunity in bacterial cells by degrading invading viral genomes. By democratizing this activity into human cells, it is possible to knock out specific genes to disable their function and repair errors. The latter of these activities requires the participation of a single-stranded donor DNA template that provides the genetic information to execute correction in a process referred to as homology directed repair (HDR). Here, we utilized an established cell-free extract system to determine the influence that the donor DNA template length has on the diversity of products from CRISPR-directed gene editing. This model system enables us to view all outcomes of this reaction and reveals that donor template length can influence the efficiency of the reaction and the categories of error-prone products that accompany it. A careful measurement of the products revealed a category of error-prone events that contained the corrected template along with insertions and deletions (indels). Our data provides foundational information for those whose aim is to translate CRISPR/Cas from bench to bedside.

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