scholarly journals Precise genome engineering inDrosophilausing prime editing

2020 ◽  
Vol 118 (1) ◽  
pp. e2021996118
Author(s):  
Justin A. Bosch ◽  
Gabriel Birchak ◽  
Norbert Perrimon
Keyword(s):  
Gene Function ◽  
Mammalian Cells ◽  
Genome Engineering ◽  
Cultured Cells ◽  
Germ Line ◽  
Point Mutations ◽  
Model Organism ◽  
Model Organisms ◽  
Marker Genes ◽  
Germ Line Transmission

Precise genome editing is a valuable tool to study gene function in model organisms. Prime editing, a precise editing system developed in mammalian cells, does not require double-strand breaks or donor DNA and has low off-target effects. Here, we applied prime editing for the model organismDrosophila melanogasterand developed conditions for optimal editing. By expressing prime editing components in cultured cells or somatic cells of transgenic flies, we precisely introduce premature stop codons in three classical visible marker genes,ebony,white, andforked. Furthermore, by restricting editing to germ cells, we demonstrate efficient germ-line transmission of a precise edit inebonyto 36% of progeny. Our results suggest that prime editing is a useful system inDrosophilato study gene function, such as engineering precise point mutations, deletions, or epitope tags.

scholarly journals Precise genome engineering in Drosophila using prime editing

2020 ◽  
Author(s):  
Justin A. Bosch ◽  
Gabriel Birchak ◽  
Norbert Perrimon
Keyword(s):  
Gene Function ◽  
Mammalian Cells ◽  
Genome Engineering ◽  
Cultured Cells ◽  
Germ Line ◽  
Point Mutations ◽  
Model Organism ◽  
Model Organisms ◽  
Marker Genes ◽  
Germ Line Transmission

AbstractPrecise genome editing is a valuable tool to study gene function in model organisms. Prime editing, a precise editing system developed in mammalian cells, does not require double strand breaks or donor DNA and has low off-target effects. Here, we applied prime editing for the model organism Drosophila melanogaster and developed conditions for optimal editing. By expressing prime editing components in cultured cells or somatic cells of transgenic flies, we precisely installed premature stop codons in three classical visible marker genes, ebony, white, and forked. Furthermore, by restricting editing to germ cells, we demonstrate efficient germ line transmission of a precise edit in ebony to ~50% of progeny. Our results suggest that prime editing is a useful system in Drosophila to study gene function, such as engineering precise point mutations, deletions, or epitope tags.

scholarly journals Genome-engineering with CRISPR-Cas9 in the mosquitoAedes aegypti

2014 ◽  
Author(s):  
Kathryn E Kistler ◽  
Leslie B Vosshall ◽  
Benjamin J Matthews
Keyword(s):  
Genome Engineering ◽  
Germ Line ◽  
Dna Binding Protein ◽  
Model Organisms ◽  
Homing Endonucleases ◽  
Loss Of Function ◽  
Dna Base ◽  
Different Types ◽  
Flexible Genome ◽  
Repair Mechanisms

The mosquitoAedes aegyptiis a potent vector of the Chikungunya, yellow fever, and Dengue viruses, which result in hundreds of millions of infections and over 50,000 human deaths per year. Loss-of-function mutagenesis inAe. aegyptihas been established with TALENs, ZFNs, and homing endonucleases, which require the engineering of DNA-binding protein domains to generate target specificity for a particular stretch of genomic DNA. Here, we describe the first use of the CRISPR-Cas9 system to generate targeted, site-specific mutations inAe. aegypti. CRISPR-Cas9 relies on RNA-DNA base-pairing to generate targeting specificity, resulting in cheaper, faster, and more flexible genome-editing reagents. We investigate the efficiency of reagent concentrations and compositions, demonstrate the ability of CRISPR-Cas9 to generate several different types of mutations via disparate repair mechanisms, and show that stable germ-line mutations can be readily generated at the vast majority of genomic loci tested. This work offers a detailed exploration into the optimal use of CRISPR-Cas9 inAe. aegyptithat should be applicable to non-model organisms previously out of reach of genetic modification.

scholarly journals Establishment of CRISPR/Cas9-based knock-in in a hemimetabolous insect: targeted gene tagging in the cricket Gryllus bimaculatus

2021 ◽  
Author(s):  
Yuji Matsuoka ◽  
Taro Nakamura ◽  
Takahito Watanabe ◽  
Austen A. Barnett ◽  
Sumihare Noji ◽  
...  
Keyword(s):  
Germ Line ◽  
Fruit Fly ◽  
Model Organisms ◽  
Induced Mutations ◽  
Gryllus Bimaculatus ◽  
Knock Out ◽  
Fluorescent Marker ◽  
Hemimetabolous Insect ◽  
Targeted Nucleases ◽  
Germ Line Transmission

Studies of traditional model organisms like the fruit fly Drosophila melanogaster have contributed immensely to our understanding of the genetic basis of developmental processes. However, the generalizability of these findings cannot be confirmed without functional genetic analyses in additional organisms. Direct genome editing using targeted nucleases has the potential to transform hitherto poorly-understood organisms into viable laboratory organisms for functional genetic study. To this end, here we present a method to induce targeted genome knock-out and knock-in of desired sequences in an insect that serves as an informative contrast to Drosophila, the cricket Gryllus bimaculatus. The efficiency of germ line transmission of induced mutations is comparable to that reported for other well-studied laboratory organisms, and knock-ins targeting introns yields viable, fertile animals in which knock-in events are directly detectable by visualization of a fluorescent marker in the expression pattern of the targeted gene. Combined with the recently assembled and annotated genome of this cricket, this knock-in/knock-out method increases the viability of G. bimaculatus as a tractable system for functional genetics in a basally branching insect.

Inheritance of phenotype in mammalian cells: genetic vs. epigenetic mechanisms

1991 ◽  
Vol 260 (6) ◽  
pp. L386-L394 ◽  
Author(s):  
D. C. Gruenert ◽  
A. L. Cozens
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Growth Regulation ◽  
Cultured Cells ◽  
Protein Complexes ◽  
Point Mutations ◽  
Genetic Alterations ◽  
Epigenetic Mechanisms ◽  
Phenotypic Changes ◽  
Pathways Analysis

Inherited phenotypic changes in cultured cells, as observed during differentiation and transformation, reflect alterations in gene expression and have both a genetic and epigenetic basis. The causes of specific changes are often difficult to define especially when observing phenomenological end points. Although such observations are an important step in defining the phenotypic changes that endure for multiple generations, it is necessary to analyze cells at the molecular level to characterize the pathways leading to changes in phenotype. Gene expression can be regulated at multiple levels, i.e., DNA structure, gene transcription, and/or posttranscriptional modifications. Four genetic mechanisms (DNA point mutations, deletion, rearrangement, and amplification) and two epigenetic mechanisms (DNA methylation and the preservation of DNA-protein complexes) can account for the majority of enduring changes observed in cultured cells. Genetic alterations in DNA sequence appear to be largely responsible for altered growth regulation associated with transformation, but there is also evidence to suggest that epigenetic mechanisms play a role in transformation. Differentiation of cultured cells is often associated with lack of growth, and has been ascribed in part to epigenetic mechanisms. However, differentiation and transformation are not mutually exclusive but may be regulated by parallel multistep pathways. Analysis of the causes underlying transformation has been complicated by the use of aneuploid cells, but it is clear that the tools for overcoming the ambiguities associated with phenomenological analysis are available.

scholarly journals Detection of CRISPR-Cas9-Mediated Mutations Using a Carbon Nanotube-Modified Electrochemical Genosensor

Biosensors ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 17
Author(s):  
Ezgi Kivrak ◽  
Tekle Pauzaite ◽  
Nikki A. Copeland ◽  
John G. Hardy ◽  
Pinar Kara ◽  
...  
Keyword(s):  
Carbon Nanotube ◽  
Gene Editing ◽  
Genome Engineering ◽  
Point Mutations ◽  
Differential Pulse ◽  
Model Organisms ◽  
Target Sequence ◽  
Label Free ◽  
Graphite Electrodes ◽  
Guanine Oxidation

The CRISPR-Cas9 system has facilitated the genetic modification of various model organisms and cell lines. The outcomes of any CRISPR-Cas9 assay should be investigated to ensure/improve the precision of genome engineering. In this study, carbon nanotube-modified disposable pencil graphite electrodes (CNT/PGEs) were used to develop a label-free electrochemical nanogenosensor for the detection of point mutations generated in the genome by using the CRISPR-Cas9 system. Carbodiimide chemistry was used to immobilize the 5′-aminohexyl-linked inosine-substituted probe on the surface of the sensor. After hybridization between the target sequence and probe at the sensor surface, guanine oxidation signals were monitored using differential pulse voltammetry (DPV). Optimization of the sensitivity of the nanogenoassay resulted in a lower detection limit of 213.7 nM. The nanogenosensor was highly specific for the detection of the precisely edited DNA sequence. This method allows for a rapid and easy investigation of the products of CRISPR-based gene editing and can be further developed to an array system for multiplex detection of different-gene editing outcomes.

scholarly journals Peel-1 negative selection promotes screening-free CRISPR-Cas9 genome editing in Caenorhabditis elegans

2020 ◽  
Author(s):  
Troy A. McDiarmid ◽  
Vinci Au ◽  
Donald G. Moerman ◽  
Catharine H. Rankin
Keyword(s):  
Caenorhabditis Elegans ◽  
Machine Vision ◽  
Negative Selection ◽  
Large Scale ◽  
Genome Engineering ◽  
Model Organism ◽  
Model Organisms ◽  
Behavioral Phenotypes ◽  
Functional Studies ◽  
Marker Selection

AbstractImproved genome engineering methods that enable automation of large and precise edits are essential for systematic investigations of genome function. We adapted peel-1 negative selection to an optimized Dual-Marker Selection (DMS) cassette protocol for CRISPR-Cas9 genome engineering in Caenorhabditis elegans and observed robust increases in multiple measures of efficiency that were consistent across injectors and four genomic loci. The use of Peel-1-DMS selection killed animals harboring transgenes as extrachromosomal arrays and spared genome edited integrants, often circumventing the need for visual screening to identify genome edited animals. To demonstrate the applicability of the approach, we created deletion alleles in the putative proteasomal subunit pbs-1 and the uncharacterized gene K04F10.3 and used machine vision to automatically characterize their phenotypic profiles, revealing homozygous essential and heterozygous behavioral phenotypes. These results provide a robust and scalable approach to rapidly generate and phenotype genome edited animals without the need for screening or scoring by eye.Author summaryThe ability to directly manipulate the genome and observe the resulting effects on the traits of an organism is a powerful approach to investigate gene function. CRISPR-based approaches to genome engineering have revolutionized such functional studies across model organisms but still face major challenges that limit the scope and complexity of projects that can be achieved in practice. Automating genome engineering and phenotyping would enable large-scale investigations of genome function in animals. Here, we describe the adaptation of peel-1 negative selection to an optimized dual-marker selection cassette CRISPR-Cas9 genome engineering method in C. elegans and combine it with automated machine vision phenotyping to achieve functional studies without the need for screening or scoring by eye. To demonstrate the applicability of the approach, we generated novel deletion alleles in two understudied genes, pbs-1 and K04F10.3, and used machine vision to characterize their phenotypic profiles, revealing homozygous lethal and heterozygous behavioral phenotypes. Our results open the door to systematic investigations of genome function in this model organism.

scholarly journals The Zebrafish Information Network: major gene page and home page updates

2020 ◽  
Vol 49 (D1) ◽  
pp. D1058-D1064 ◽  
Author(s):  
Douglas G Howe ◽  
Sridhar Ramachandran ◽  
Yvonne M Bradford ◽  
David Fashena ◽  
Sabrina Toro ◽  
...  
Keyword(s):  
Gene Function ◽  
Major Gene ◽  
Model Organism ◽  
Home Page ◽  
Model Organisms ◽  
Disease Models ◽  
Information Network ◽  
Similar Data ◽  
Mutant Phenotypes ◽  
Gene Page

Abstract The Zebrafish Information Network (ZFIN) (https://zfin.org/) is the database for the model organism, zebrafish (Danio rerio). ZFIN expertly curates, organizes, and provides a wide array of zebrafish genetic and genomic data, including genes, alleles, transgenic lines, gene expression, gene function, mutant phenotypes, orthology, human disease models, gene and mutant nomenclature, and reagents. New features at ZFIN include major updates to the home page and the gene page, the two most used pages at ZFIN. Data including disease models, phenotypes, expression, mutants and gene function continue to be contributed to The Alliance of Genome Resources for integration with similar data from other model organisms.

scholarly journals The fruit fly at the interface of diagnosis and pathogenic mechanisms of rare and common human diseases

2019 ◽  
Vol 28 (R2) ◽  
pp. R207-R214 ◽  
Author(s):  
Hugo J Bellen ◽  
Michael F Wangler ◽  
Shinya Yamamoto
Keyword(s):  
Rare Disease ◽  
Gene Function ◽  
Rare Variants ◽  
Genetic Model ◽  
Model Organism ◽  
Model Organisms ◽  
Pathogenic Mechanisms ◽  
Disease Mechanisms ◽  
The Impact ◽  
Undiagnosed Diseases

Abstract Drosophila melanogaster is a unique, powerful genetic model organism for studying a broad range of biological questions. Human studies that probe the genetic causes of rare and undiagnosed diseases using massive-parallel sequencing often require complementary gene function studies to determine if and how rare variants affect gene function. These studies also provide inroads to disease mechanisms and therapeutic targets. In this review we discuss strategies for functional studies of rare human variants in Drosophila. We focus on our experience in establishing a Drosophila core of the Model Organisms Screening Center for the Undiagnosed Diseases Network (UDN) and concurrent fly studies with other large genomic rare disease research efforts such as the Centers for Mendelian Genomics. We outline four major strategies that use the latest technology in fly genetics to understand the impact of human variants on gene function. We also mention general concepts in probing disease mechanisms, therapeutics and using rare disease to understand common diseases. Drosophila is and will continue to be a fundamental genetic model to identify new disease-causing variants, pathogenic mechanisms and drugs that will impact medicine.

scholarly journals ESCargo: a regulatable fluorescent secretory cargo for diverse model organisms

2020 ◽  
Author(s):  
Jason C. Casler ◽  
Allison L. Zajac ◽  
Fernando M. Valbuena ◽  
Daniela Sparvoli ◽  
Okunola Jeyifous ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Signal Sequence ◽  
Model Organism ◽  
Kinetic Studies ◽  
Secretory Protein ◽  
Yeast Cells ◽  
Model Organisms ◽  
Cargo Protein ◽  
Simple Technology

AbstractMembrane traffic can be studied by imaging a cargo protein as it transits the secretory pathway. The best tools for this purpose initially block exit of the secretory cargo from the endoplasmic reticulum (ER), and then release the block to generate a cargo wave. However, previously developed regulatable secretory cargoes are often tricky to use or specific for a single model organism. To overcome these hurdles for budding yeast, we recently optimized an artificial fluorescent secretory protein that exits the ER with the aid of the Erv29 cargo receptor, which is homologous to mammalian Surf4. The fluorescent secretory protein forms aggregates in the ER lumen and can be rapidly disaggregated by addition of a ligand to generate a nearly synchronized cargo wave. Here we term this regulatable secretory protein ESCargo (Erv29/Surf4-dependent Secretory Cargo) and demonstrate its utility not only in yeast cells, but also in cultured mammalian cells, Drosophila cells, and the ciliate Tetrahymena thermophila. Kinetic studies indicate that rapid transport out of the ER requires recognition by Erv29/Surf4. By choosing an appropriate ER signal sequence and expression vector, this simple technology can likely be used with many model organisms.

scholarly journals Gene knock-ins in Drosophila using homology-independent insertion of universal donor plasmids

2019 ◽  
Author(s):  
Justin A. Bosch ◽  
Ryan Colbeth ◽  
Jonathan Zirin ◽  
Norbert Perrimon
Keyword(s):  
Gene Function ◽  
Target Gene ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Germ Line ◽  
Insertion Site ◽  
End Joining ◽  
Endogenous Proteins ◽  
Non Homologous End Joining ◽  
Universal Donor

AbstractTargeted genomic knock-ins are a valuable tool to probe gene function. However, knock-in methods involving homology-directed repair (HDR) can be laborious. Here, we adapt the mammalian CRISPaint homology-independent knock-in method for Drosophila melanogaster, which uses CRISPR/Cas9 and non-homologous end joining (NHEJ) to insert universal donor plasmids into the genome. This method is a simple and fast alternative to HDR for certain strategies such as C-terminal tagging and gene disruption. Using this method in cultured S2R+ cells, we efficiently tagged four endogenous proteins with the bright fluorescent protein mNeonGreen, thereby demonstrating that an existing collection of CRISPaint universal donor plasmids is compatible with insect cells. In addition, we inserted the transgenesis marker 3xP3-RFP into seven genes in the fly germ line, producing heritable loss of function alleles that were isolated by simple fluorescence screening. Unlike in cultured cells, indels always occurred at the genomic insertion site, which prevents predictably matching the insert coding frame to the target gene. Despite this effect, we were able to isolate T2A-Gal4 insertions in four genes that serve as in vivo expression reporters. Finally, we apply this fast knock-in method to uncharacterized small open reading frame (smORF) genes. Therefore, homology-independent insertion is a useful genome editing technique in Drosophila that will better enable researchers to dissect gene function.Article summaryWe report a fast and simple genomic knock-in method in Drosophila to insert large DNA elements into any target gene. Using CRISPR-Cas9 and non-homologous end joining (NHEJ), an entire donor plasmid is inserted into the genome without the need for homology arms. We demonstrate its usefulness in cultured cells to fluorescently tag endogenous proteins and in the fly germ line to generate heritable insertions that disrupt gene function and can act as expression reporters.

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