Transient, flexible gene editing in zebrafish neutrophils and macrophages for determination of cell-autonomous functions

ABSTRACT Zebrafish are an important model for studying phagocyte function, but rigorous experimental systems to distinguish whether phagocyte-dependent effects are neutrophil or macrophage specific have been lacking. We have developed and validated transgenic lines that enable superior demonstration of cell-autonomous neutrophil and macrophage genetic requirements. We coupled well-characterized neutrophil- and macrophage-specific Gal4 driver lines with UAS:Cas9 transgenes for selective expression of Cas9 in either neutrophils or macrophages. Efficient gene editing, confirmed by both Sanger and next-generation sequencing, occurred in both lineages following microinjection of efficacious synthetic guide RNAs into zebrafish embryos. In proof-of-principle experiments, we demonstrated molecular and/or functional evidence of on-target gene editing for several genes (mCherry, lamin B receptor, trim33) in either neutrophils or macrophages as intended. These new UAS:Cas9 tools provide an improved resource for assessing individual contributions of neutrophil- and macrophage-expressed genes to the many physiological processes and diseases modelled in zebrafish. Furthermore, this gene-editing functionality can be exploited in any cell lineage for which a lineage-specific Gal4 driver is available. This article has an associated First Person interview with the first author of the paper.

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Genetically Encoded CRISPR components Yield Efficient Gene Editing in the Invasive Pest,Drosophila suzukii

10.1101/2021.03.15.435483 ◽

2021 ◽

Author(s):

Nikolay P. Kandul ◽

Esther J. Belikoff ◽

Junru Liu ◽

Anna Buchman ◽

Fang Li ◽

...

Keyword(s):

Population Control ◽

Invasive Pest ◽

Drosophila Suzukii ◽

Guide Rnas ◽

Cas9 Nuclease ◽

Efficient Gene ◽

Transgenic Lines ◽

Repeated Applications ◽

Very High ◽

Genetic Strategies

AbstractOriginally from Asia,Drosophila suzukii(Matsumura, 1931, Diptera:Drosophilidae) is presently a global pest of economically important soft-skinned fruits. Also commonly known as spotted wingDrosophila(SWD), it is largely controlled through repeated applications of broad-spectrum insecticides. There is a pressing need for a better understanding of SWD biology and for developing alternative environmentally-friendly methods of control. The RNA-guided Cas9 nuclease has revolutionized functional genomics and is an integral component of several recently developed genetic strategies for population control of insects. Here we have developed transgenic strains that encode three different terminators and four different promoters to express Cas9 in both the soma and/or germline of SWD. The Cas9 lines were evaluated through genetic crossing to transgenic lines that encode single guide RNAs targeting the conserved X-linkedyellowbody andwhiteeye genes. We find that several Cas9/gRNA lines display very high editing capacity. Going forward, these tools will be instrumental for evaluating gene function in SWD and may provide tools useful for the development of new genetic strategies for control of this invasive species.

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RecT recombinase expression enables efficient gene editing in Enterococcus

10.1101/2020.09.01.278044 ◽

2020 ◽

Author(s):

Victor Chen ◽

Matthew G. Griffin ◽

Howard C. Hang

Keyword(s):

Enterococcus Faecium ◽

Gene Editing ◽

Specific Gene ◽

Mechanistic Studies ◽

Dna Templates ◽

Beneficial Effects ◽

Guide Rnas ◽

Genetic Components ◽

Efficient Gene ◽

Microbial Infections

AbstractEnterococcus faecium is a ubiquitous Gram-positive bacterium that has been recovered from the environment, food and microbiota of mammals. Commensal strains of E. faecium can confer beneficial effects on host physiology and immunity, but antibiotic usage has afforded antibiotic-resistant and pathogenic isolates from livestock and humans. However, the dissection of E. faecium functions and mechanisms has been restricted by inefficient gene editing methods. To address these limitations, here we report the expression of E. faecium RecT recombinase significantly improves the efficiency of recombineering technologies in commensal strains of E. faecium and other Enterococcus species such as E. durans and E. hirae. Notably, we demonstrate that E. faecium RecT expression facilitated the chromosomal insertion of both single-stranded and double-stranded DNA templates encoding antibiotic selectable markers. Moreover, the expression of RecT in combination with clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 and guide RNAs (gRNAs) enabled highly efficient scar-less ssDNA recombineering to generate specific gene editing mutants in E. faecium. The RecT-mediated recombineering methods described here should significantly enhance genetic studies of E. faecium and other closely related species for functional and mechanistic studies.ImportanceEnterococcus faecium is widely recognized as an emerging public health threat with the rise of drug resistance and nosocomial infections. Nevertheless, commensal Enterococcus strains possess beneficial health functions in mammals to upregulate host immunity and prevent microbial infections. This functional dichotomy of Enterococcus species and strains highlights the need for in-depth studies to discover and characterize the genetic components underlining its diverse activities. However, genetic engineering in E. faecium still requires passive homologous recombination, which often requires cloning of multiple homologous fragments and screening. To alleviate these challenges, we discovered that RecT-recombinase enables more efficient integration of mutagenic DNA templates to generate insertions, deletions and substitutions of genomic DNA in E. faecium. These improved recombineering methods should facilitate functional and mechanistic studies of Enterococcus.

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Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species

Journal of Fungi ◽

10.3390/jof7070505 ◽

2021 ◽

Vol 7 (7) ◽

pp. 505

Author(s):

Ping Zhang ◽

Yu Wang ◽

Chenxi Li ◽

Xiaoyu Ma ◽

Lan Ma ◽

...

Keyword(s):

Genome Editing ◽

Fungal Pathogens ◽

Gene Editing ◽

Recombination Frequency ◽

Target Sequence ◽

Widespread Application ◽

Genetic Studies ◽

Efficient Gene ◽

Cryptococcus Species ◽

Overlap Pcr

Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic fungal pathogens found worldwide that are utilized to reveal mechanisms of fungal pathogenesis. However, their low homologous recombination frequency has greatly encumbered genetic studies. In preliminary work, we described a ‘suicide’ CRISPR-Cas9 system for use in the efficient gene editing of C. deneoformans, but this has not yet been used in the C. neoformans strain. The procedures involved in constructing vectors are time-consuming, whether they involve restriction enzyme-based cloning of donor DNA or the introduction of a target sequence into the gRNA expression cassette via overlap PCR, as are sophisticated, thus impeding their widespread application. Here, we report the optimized and simplified construction method for all-in-one CRISPR-Cas9 vectors that can be used in C. neoformans and C. deneoformans strains respectively, named pNK003 (Genbank: MW938321) and pRH003 (Genbank: KX977486). Taking several gene manipulations as examples, we also demonstrate the accuracy and efficiency of the new simplified all-in-one CRISPR-Cas9 genome editing tools in both Serotype A and Serotype D strains, as well as their ability to eliminate Cas9 and gDNA cassettes after gene editing. We anticipate that the availability of new vectors that can simplify and streamline the technical steps for all-in-one CRISPR-Cas9 construction could accelerate genetic studies of the Cryptococcus species.

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Major sex-determining genes and the control of sexual dimorphism in Caenorhabditis elegans

Genome ◽

10.1139/g89-116 ◽

1989 ◽

Vol 31 (2) ◽

pp. 625-637 ◽

Cited By ~ 4

Author(s):

Jonathan Hodgkin ◽

Andrew D. Chisholm ◽

Michael M. Shen

Keyword(s):

Caenorhabditis Elegans ◽

Sex Determination ◽

X Chromosome ◽

Germ Line ◽

Cell Lineage ◽

Regulatory Genes ◽

Nematode Caenorhabditis Elegans ◽

X Chromosomes ◽

The Many ◽

Lineage Analysis

Sex determination in Caenorhabditis elegans involves a cascade of major regulatory genes connecting the primary sex determining signal, X chromosome dosage, to key switch genes, which in turn direct development along either male or female pathways. Animals with one X chromosome (XO) are male, while animals with two X chromosomes (XX) are hermaphrodite: hermaphrodite development occurs because the action of the regulatory genes is modified in the germ line so that both sperm and oocytes are made inside a completely female soma. The regulatory genes are being examined by both genetic and molecular means. We discuss how these major genes, in particular the last switch gene in the cascade, tra-1, might regulate the many different sex-specific events that occur during the development of the hermaphrodite and of the male.Key words: nematode, Caenorhabditis elegans, sex determination, sexual differentiation, cell lineage analysis.

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Design of time-delayed safety switches for CRISPR gene therapy

10.1101/2021.04.17.440285 ◽

2021 ◽

Author(s):

Dashan Sun

Keyword(s):

Gene Therapy ◽

Immune Response ◽

Time Delay ◽

Cell Lines ◽

Gene Editing ◽

Drug Accumulation ◽

Work Time ◽

Crispr System ◽

Efficient Gene ◽

Target Effect

CRISPR system is a powerful gene editing tool which has already been reported to address a variety of gene relevant diseases in different cell lines. However, off-target effect and immune response caused by Cas9 remain two fundamental problems. In our work, time-delayed safety switches are designed based on either artificial ultrasensitivity transmission module or intrinsic time delay in biomolecular activities. By addressing gene therapy efficiency, off-target effect, immune response and drug accumulation, we hope our safety switches may offer inspiration in realizing safe and efficient gene therapy in humans.

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Cas9 conjugate complex delivering donor DNA for efficient gene editing by homology-directed repair

Journal of Industrial and Engineering Chemistry ◽

10.1016/j.jiec.2021.07.009 ◽

2021 ◽

Author(s):

Yoo Kyung Kang ◽

Ju Hee Lee ◽

San Hae Im ◽

Joo Hoon Lee ◽

Juhee Jeong ◽

...

Keyword(s):

Gene Editing ◽

Homology Directed Repair ◽

Efficient Gene

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714. Structure-Based Engineering of the FokI DNA Cleavage Domain in the Context of a Chimeric Endonuclease: Towards Maximally Efficient “Gene Editing” Therapy

Molecular Therapy ◽

10.1016/j.ymthe.2004.06.620 ◽

2004 ◽

Vol 9 ◽

pp. S272

Keyword(s):

Dna Cleavage ◽

Gene Editing ◽

Efficient Gene ◽

Cleavage Domain

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Exploration of CRISPR/Cas9-based gene editing as therapy for osteoarthritis

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2018-214724 ◽

2019 ◽

Vol 78 (5) ◽

pp. 676-682 ◽

Cited By ~ 22

Author(s):

Lan Zhao ◽

Jian Huang ◽

Yunshan Fan ◽

Jun Li ◽

Tianming You ◽

...

Keyword(s):

Structural Damage ◽

Drug Targets ◽

Gene Editing ◽

Joint Damage ◽

Loss Of Function ◽

Adeno Associated Virus ◽

Structural Deterioration ◽

Efficient Gene ◽

Genes Encoding ◽

Surgically Induced

ObjectivesOsteoarthritis (OA) is a painful and debilitating disease and it is associated with aberrant upregulation of multiple factors, including matrix metalloproteinase 13 (MMP13), interleukin-1β (IL-1β) and nerve growth factor (NGF). In this study, we aimed to use the CRISPR/Cas9 technology, a highly efficient gene-editing tool, to study whether the ablation of OA-associated genes has OA-modifying effects.MethodsWe performed intra-articular injection of adeno-associated virus, which expressed CRISPR/Cas9 components to target each of the genes encoding MMP13, IL-1β and NGF, in a surgically induced OA mouse model. We also tested triple ablations of NGF, MMP13 and IL-1β.ResultsLoss-of-function of NGF palliates pain but worsens joint damage in the surgically induced OA model. Ablation of MMP13 or IL-1β reduces the expression of cartilage-degrading enzymes and attenuates structural deterioration. Targeting both MMP13 and IL-1β significantly mitigates the adverse effects of NGF blockade on the joints.ConclusionsCRISPR-mediated ablation of NGF alleviates OA pain, and deletion of MMP13-1β or IL-1β attenuates structural damage in a post-traumatic OA model. Multiplex ablations of NGF, MMP13 and IL-1β provide benefits on both pain management and joint structure maintenance. Our results suggest that CRISPR-based gene editing is useful for the identification of promising drug targets and the development of feasible therapeutic strategies for OA treatment.

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