scholarly journals RecT recombinase expression enables efficient gene editing in Enterococcus

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.

scholarly journals RecT recombinase expression enables efficient gene editing in Enterococcus

2021 ◽  
Author(s):  
Victor Chen ◽  
Matthew E. Griffin ◽  
Pascal Maguin ◽  
Andrew Varble ◽  
Howard C. Hang
Keyword(s):  
Enterococcus Faecium ◽  
Gene Editing ◽  
Functional Characterization ◽  
Specific Gene ◽  
Mechanistic Studies ◽  
Dna Templates ◽  
Antibiotic Resistant ◽  
Knock Out ◽  
Beneficial Effects ◽  
Genetic Components

Enterococcus 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 both commensal and antibiotic-resistant strains of E. faecium and other Enterococcus species such as E. durans and E. hirae . Notably, the expression of RecT in combination with clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 and guide RNAs (gRNAs) enabled highly efficient scar-less single-stranded DNA recombineering to generate specific gene editing mutants in E. faecium . Moreover, we demonstrate that E. faecium RecT expression facilitated chromosomal insertions of double-stranded DNA templates encoding antibiotic selectable markers to generate gene deletion mutants. As further proof-of-principle, we use CRISPR-Cas9 mediated recombineering to knock out both sortase A genes in E. faecium for downstream functional characterization. The general RecT-mediated recombineering methods described here should significantly enhance genetic studies of E. faecium and other closely related species for functional and mechanistic studies. Importance Enterococcus 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, current genetic engineering methods in E. faecium still require passive homologous recombination from plasmid DNA. This involves the successful cloning of multiple homologous fragments into a plasmid, introducing the plasmid into E. faecium , and screening for double-crossover events that can collectively take up to multiple weeks to perform. To alleviate these challenges, we show that RecT recombinase enables rapid and efficient integration of mutagenic DNA templates to generate substitutions, deletions, and insertions in genomic DNA of E. faecium . These improved recombineering methods should facilitate functional and mechanistic studies of Enterococcus .

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

2021 ◽  
Vol 14 (7) ◽  
Author(s):  
Abdulsalam I. Isiaku ◽  
Zuobing Zhang ◽  
Vahid Pazhakh ◽  
Harriet R. Manley ◽  
Ella R. Thompson ◽  
...  
Keyword(s):  
Gene Editing ◽  
Cell Lineage ◽  
Gal4 Driver ◽  
Guide Rnas ◽  
Experimental Systems ◽  
Physiological Processes ◽  
Lamin B Receptor ◽  
Efficient Gene ◽  
Transgenic Lines ◽  
The Many

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.

scholarly journals New and TALENted Genome Engineering Toolbox

2013 ◽  
Vol 113 (5) ◽  
pp. 571-587 ◽  
Author(s):  
Jarryd M. Campbell ◽  
Katherine A. Hartjes ◽  
Timothy J. Nelson ◽  
Xiaolei Xu ◽  
Stephen C. Ekker
Keyword(s):  
Cardiovascular Disease ◽  
Gene Editing ◽  
Genome Engineering ◽  
Genetic Alterations ◽  
Model Systems ◽  
Patient Specific ◽  
Specific Gene ◽  
Genetic Defects ◽  
Throughput Model ◽  
Efficient Gene

Recent advances in the burgeoning field of genome engineering are accelerating the realization of personalized therapeutics for cardiovascular disease. In the postgenomic era, sequence-specific gene-editing tools enable the functional analysis of genetic alterations implicated in disease. In partnership with high-throughput model systems, efficient gene manipulation provides an increasingly powerful toolkit to study phenotypes associated with patient-specific genetic defects. Herein, this review emphasizes the latest developments in genome engineering and how applications within the field are transforming our understanding of personalized medicine with an emphasis on cardiovascular diseases.

scholarly journals Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

Biology ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 530
Author(s):  
Marlo K. Thompson ◽  
Robert W. Sobol ◽  
Aishwarya Prakash
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Gene Editing ◽  
Adaptive Immune System ◽  
Zinc Finger Nucleases ◽  
Specific Gene ◽  
Target Sequence ◽  
Transcription Activator ◽  
Low Cytotoxicity

The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.

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

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.

High-throughput vectors for efficient gene silencing in plants

2002 ◽  
Vol 29 (10) ◽  
pp. 1217 ◽  
Author(s):  
Chris A. Helliwell ◽  
S. Varsha Wesley ◽  
Anna J. Wielopolska ◽  
Peter M. Waterhouse
Keyword(s):  
Gene Silencing ◽  
Insertional Mutagenesis ◽  
Single Step ◽  
Plant Genome ◽  
Specific Gene ◽  
Single Polymerase Chain Reaction ◽  
Efficient Gene ◽  
Gene Redundancy ◽  
Endogenous Genes ◽  
Rapid Generation

A major challenge in the post-genome era of plant biology is to determine the functions of all genes in the plant genome. A straightforward approach to this problem is to reduce or knockout expression of a gene with the hope of seeing a phenotype that is suggestive of its function. Insertional mutagenesis is a useful tool for this type of study but is limited by gene redundancy, lethal knockouts, non-tagged mutants, and the inability to target the inserted element to a specific gene. The efficacy of gene silencing in plants using inverted-repeat transgene constructs that encode a hairpin RNA (hpRNA) has been demonstrated by a number of groups, and has several advantages over insertional mutagenesis. In this paper we describe two improved pHellsgate vectors that facilitate rapid generation of hpRNA-encoding constructs. pHellsgate 4 allows the production of an hpRNA construct in a single step from a single polymerase chain reaction product, while pHellsgate 8 requires a two-step process via an intermediate vector. We show that these vectors are effective at silencing three endogenous genes in Arabidopsis, FLOWERING LOCUS C, PHYTOENE DESATURASE and ETHYLENE INSENSITIVE 2. We also show that a construct of sequences from two genes silences both genes.

scholarly journals Allele-specific gene editing rescues pathology in a human model of Charcot-Marie-Tooth disease type 2E

2021 ◽  
Author(s):  
Carissa M. Feliciano ◽  
Kenneth Wu ◽  
Hannah L. Watry ◽  
Chiara B.E. Marley ◽  
Gokul N. Ramadoss ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Light Chain ◽  
Gene Editing ◽  
Human Model ◽  
Specific Gene ◽  
Neurofilament Light Chain ◽  
Neurofilament Light ◽  
Light Chain Gene ◽  
Charcot Marie Tooth ◽  
Allele Specific

Many neuromuscular disorders are caused by dominant missense mutations that lead to dominant-negative or gain-of-function pathology. This category of disease is challenging to address via drug treatment or gene augmentation therapy because these strategies may not eliminate the effects of the mutant protein or RNA. Thus, effective treatments are severely lacking for these dominant diseases, which often cause severe disability or death. The targeted inactivation of dominant disease alleles by gene editing is a promising approach with the potential to completely remove the cause of pathology with a single treatment. Here, we demonstrate that allele-specific CRISPR gene editing in a human model of axonal Charcot-Marie-Tooth (CMT) disease rescues pathology caused by a dominant missense mutation in the neurofilament light chain gene (NEFL, CMT type 2E). We utilized a rapid and efficient method for generating spinal motor neurons from human induced pluripotent stem cells (iPSCs) derived from a patient with CMT2E. Diseased motor neurons recapitulated known pathologic phenotypes at early time points of differentiation, including aberrant accumulation of neurofilament light chain protein in neuronal cell bodies. We selectively inactivated the disease NEFL allele in patient iPSCs using Cas9 enzymes to introduce a frameshift at the pathogenic N98S mutation. Motor neurons carrying this allele-specific frameshift demonstrated an amelioration of the disease phenotype comparable to that seen in an isogenic control with precise correction of the mutation. Our results validate allele-specific gene editing as a therapeutic approach for CMT2E and as a promising strategy to silence dominant mutations in any gene for which heterozygous loss-of-function is well tolerated. This highlights the potential for gene editing as a therapy for currently untreatable dominant neurologic diseases.

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

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.

scholarly journals Allele-specific gene editing to rescue dominant CRX-associated LCA7 phenotypes in a retinal organoid model

2021 ◽  
Author(s):  
Kathleen R. Chirco ◽  
Shereen Chew ◽  
Anthony T. Moore ◽  
Jacque L. Duncan ◽  
Deepak A. Lamba
Keyword(s):  
Gene Editing ◽  
Specific Gene ◽  
Allele Specific

scholarly journals Design of time-delayed safety switches for CRISPR gene therapy

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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