Knock-in and Knock-out: The Use of Reverse Genetics in Somatic Cells to Dissect Mitotic Pathways

Normal gonad development assures the fertility of the individual. The properly functioning gonads must contain a sufficient number of the viable germ cells, possess a correct architecture and tissue structure, and assure the proper hormonal regulation. This is achieved by the interplay between the germ cells and different types of somatic cells. N-cadherin coded by the Cdh2 gene plays a critical role in this interplay. To gain an insight into the role of N-cadherin in the development of mouse gonads, we used the Cre-loxP system to knock out N-cadherin separately in two cell lines: the SF1+ somatic cells and the OCT4+ germ cells. We observed that N-cadherin plays a key role in the survival of both female and male germ cells. However, the N-cadherin is not necessary for the differentiation of the Sertoli cells or the initiation of the formation of testis cords or ovigerous cords. In the later stages of gonad development, N-cadherin is important for the maintenance of testis cord structure and is required for the formation of steroidogenic cells. In the ovaries, N-cadherin is necessary for the formation of the ovarian follicles. These results indicate that N-cadherin plays a major role in gonad differentiation, structuralization, and function.

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A dual sgRNA approach for functional genomics in Arabidopsis thaliana[OPEN]

10.1101/172676 ◽

2017 ◽

Author(s):

Laurens Pauwels ◽

Rebecca De Clercq ◽

Jonas Goossens ◽

Sabrina Iñigo ◽

Clara Williams ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Gene Editing ◽

Reverse Genetics ◽

Gene Families ◽

Null Alleles ◽

Loss Of Function ◽

Knock Out ◽

Nonsense Mediated Decay ◽

Dna Insertion ◽

Entire Gene

AbstractReverse genetics uses loss-of-function alleles to interrogate gene function. The advent of CRISPR/Cas9-based gene editing now allows to generate knock-out alleles for any gene and entire gene families. Even in the model plant Arabidopsis thaliana, gene editing is welcomed as T-DNA insertion lines do not always generate null alleles. Here, we show efficient generation of heritable mutations in Arabidopsis using CRISPR/Cas9 with a workload similar to generating overexpression lines. We obtain Cas9 null-segregants with bi-allelic mutations in the T2 generation. Out of seven new mutant alleles we report here, one allele for GRXS17, the ortholog of human GRX3/PICOT, did not phenocopy previously characterized nulls. Notwithstanding, the mutation caused a frameshift and triggered nonsense-mediated decay. We demonstrate that our workflow is also compatible with a dual sgRNA approach in which a gene is targeted by two sgRNAs simultaneously. This paired nuclease method can result in a more reliable loss-of-function alleles that lack a large essential part of the gene. The ease in the CRISPR/Cas9 workflow should help in the eventual generation of true null alleles of every gene in the Arabidopsis genome, which will advance both basic and applied plant research.One-sentence summaryWe present a dual sgRNA approach to delete Arabidopsis gene 34 fragments in order to obtain reliable functional knock-outs.

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N-glycosylation of infectious bronchitis virus M41 spike determines receptor specificity

Journal of General Virology ◽

10.1099/jgv.0.001408 ◽

2020 ◽

Vol 101 (6) ◽

pp. 599-608

Author(s):

K. M. Bouwman ◽

N. Habraeken ◽

A. Laconi ◽

A. J. Berends ◽

L. Groenewoud ◽

...

Keyword(s):

Infectious Bronchitis Virus ◽

Reverse Genetics ◽

Glycosylation Site ◽

Host Cells ◽

Receptor Specificity ◽

Knock Out ◽

Recombinant Viruses ◽

Infectious Bronchitis

Infection of chicken coronavirus infectious bronchitis virus (IBV) is initiated by binding of the viral heavily N-glycosylated attachment protein spike to the alpha-2,3-linked sialic acid receptor Neu5Ac. Previously, we have shown that N-glycosylation of recombinantly expressed receptor binding domain (RBD) of the spike of IBV-M41 is of critical importance for binding to chicken trachea tissue. Here we investigated the role of N-glycosylation of the RBD on receptor specificity and virus replication in the context of the virus particle. Using our reverse genetics system we were able to generate recombinant IBVs for nine-out-of-ten individual N-glycosylation mutants. In vitro growth kinetics of these viruses were comparable to the virus containing the wild-type M41-S1. Furthermore, Neu5Ac binding by the recombinant viruses containing single N-glycosylation site knock-out mutations matched the Neu5Ac binding observed with the recombinant RBDs. Five N-glycosylation mutants lost the ability to bind Neu5Ac and gained binding to a different, yet unknown, sialylated glycan receptor on host cells. These results demonstrate that N-glycosylation of IBV is a determinant for receptor specificity.

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Construction of Multiple Guide RNAs in CRISPR/Cas9 Vector Using Stepwise or Simultaneous Golden Gate Cloning: Case Study for Targeting the FAD2 and FATB Multigene in Soybean

Plants ◽

10.3390/plants10112542 ◽

2021 ◽

Vol 10 (11) ◽

pp. 2542

Author(s):

Won-Nyeong Kim ◽

Hye-Jeong Kim ◽

Young-Soo Chung ◽

Hyun-Uk Kim

Keyword(s):

Reverse Genetics ◽

Restriction Enzymes ◽

Golden Gate ◽

Knock Out ◽

Guide Rnas ◽

Genetics Research ◽

Multiple Genes ◽

Cas9 Protein ◽

Cloning Method ◽

Golden Gate Cloning

CRISPR/Cas9 is a commonly used technique in reverse-genetics research to knock out a gene of interest. However, when targeting a multigene family or multiple genes, it is necessary to construct a vector with multiple single guide RNAs (sgRNAs) that can navigate the Cas9 protein to the target site. In this protocol, the Golden Gate cloning method was used to generate multiple sgRNAs in the Cas9 vector. The vectors used were pHEE401E_UBQ_Bar and pBAtC_tRNA, which employ a one-promoter/one-sgRNA and a polycistronic-tRNA-gRNA strategy, respectively. Golden Gate cloning was performed with type IIS restriction enzymes to generate gRNA polymers for vector inserts. Four sgRNAs containing the pHEE401E_UBQ_Bar vector and four to six sgRNAs containing the pBAtC_tRNA vector were constructed. In practice, we constructed multiple sgRNAs targeting multiple genes of FAD2 and FATB in soybean using this protocol. These three vectors were transformed into soybeans using the Agrobacterium-mediated method. Using deep sequencing, we confirmed that the T0 generation transgenic soybean was edited at various indel ratios in the predicted target regions of the FAD2 and FATB multigenes. This protocol is a specific guide that allows researchers to easily follow the cloning of multiple sgRNAs into commonly used CRISPR/Cas9 vectors for plants.

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Development and Characterization of an Ethyl Methane Sulfonate (EMS) Induced Mutant Population in Capsicum annuum L.

Plants ◽

10.3390/plants9030396 ◽

2020 ◽

Vol 9 (3) ◽

pp. 396

Author(s):

Muhammad Irfan Siddique ◽

Seungki Back ◽

Joung-Ho Lee ◽

Jinkwan Jo ◽

Siyoung Jang ◽

...

Keyword(s):

Capsicum Annuum ◽

Reverse Genetics ◽

Growth Habit ◽

Ethyl Methane Sulfonate ◽

Induced Mutations ◽

Methane Sulfonate ◽

Ethyl Methane ◽

Mutant Population ◽

Knock Out ◽

Plant Growth Habit

Plant breeding explores genetic diversity in useful traits to develop new, high-yielding, and improved cultivars. Ethyl methane sulfonate (EMS) is a chemical widely used to induce mutations at loci that regulate economically essential traits. Additionally, it can knock out genes, facilitating efforts to elucidate gene functions through the analysis of mutant phenotypes. Here, we developed a mutant population using the small and pungent ornamental Capsicum annuum pepper “Micro-Pep”. This accession is particularly suitable for mutation studies and molecular research due to its compact growth habit and small size. We treated 9500 seeds with 1.3% EMS and harvested 3996 M2 lines. We then selected 1300 (32.5%) independent M2 families and evaluated their phenotypes over four years. The mutants displayed phenotypic variations in plant growth, habit, leaf color and shape, and flower and fruit morphology. An experiment to optimize Targeting Induced Local Lesions IN Genomes (TILLING) in pepper detected nine EMS-induced mutations in the eIF4E gene. The M2 families developed here exhibited broad phenotypic variation and should be valuable genetic resources for functional gene analysis in pepper molecular breeding programs using reverse genetics tools, including TILLING.

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Targeted mutation of barley (1,3;1,4)-β-glucan synthases reveals complex relationships between the storage and cell wall polysaccharide content

10.1101/2020.05.13.093146 ◽

2020 ◽

Author(s):

Guillermo Garcia-Gimenez ◽

Abdellah Barakate ◽

Pauline Smith ◽

Jennifer Stephens ◽

Shi F. Khor ◽

...

Keyword(s):

Cell Wall ◽

Reverse Genetics ◽

Grain Morphology ◽

Single Amino Acid ◽

Cell Wall Polysaccharide ◽

Thousand Grain Weight ◽

Knock Out ◽

Targeted Mutation ◽

Length Width ◽

Complex Relationships

SummaryBarley (Hordeum vulgare L) grain is comparatively rich in (1,3;1,4)-β-glucan, a source of fermentable dietary fibre that protects against various human health conditions. However, low grain (1,3;1,4)-β-glucan content is preferred for brewing and distilling. We took a reverse genetics approach, using CRISPR/Cas9 to generate mutations in members of the Cellulose synthase-like (Csl) gene superfamily that encode known (HvCslF6 and HvCslH1) and putative (HvCslF3 and HvCslF9) (1,3;1,4)-β-glucan synthases. Resultant mutations ranged from single amino acid (aa) substitutions to frameshift mutations causing premature stop codons, and led to specific differences in grain morphology, composition and (1,3;1,4)-β-glucan content. (1,3;1,4)-β-Glucan was absent in the grain of cslf6 knock-out lines whereas cslf9 knock-out lines had similar (1,3;1,4)-β-glucan content to WT. However, cslf9 mutants showed changes in the abundance of other cell wall-related monosaccharides compared to WT. Thousand grain weight (TGW), grain length, width and surface area were altered in cslf6 knock-outs and to a lesser extent TGW in cslf9 knock-outs. cslf3 and cslh1 mutants had no effect on grain (1,3;1,4)-β-glucan content. Our data indicate that multiple members of the CslF/H family fulfil important functions during grain development but, with the exception of HvCslF6, do not impact the abundance of (1,3;1,4)-β-glucan in mature grain.

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Reverse Genetic Strategies inCaenorhabditis elegans: Towards Controlled Manipulation of the Genome

The Scientific World JOURNAL ◽

10.1100/tsw.2011.126 ◽

2011 ◽

Vol 11 ◽

pp. 1394-1410 ◽

Cited By ~ 3

Author(s):

Howard A. Baylis ◽

Rafael P. Vázquez-Manrique

Keyword(s):

Gene Targeting ◽

Reverse Genetics ◽

Random Mutagenesis ◽

Zinc Finger Nucleases ◽

Gene Modification ◽

Knock Out ◽

C Elegans ◽

Endogenous Genes ◽

Endogenous Loci ◽

Genetic Strategies

Caenorhabditis eleganshas a complete annotated genome sequence that is augmented by increasing quantities of data from high-throughput postgenomic analyses. This has led to an increasing need to identify the biological functions of specific genes using reverse genetics, i.e., moving from gene to phenotype. Fundamental to this aim is the ability to alter the structure of particular genes by means that are not accessible to classical genetic strategies. Thus, one dream ofC. elegansresearchers is to establish a toolkit for the controlled manipulation of anylociwithin the genome. AlthoughC. elegansis amenable to a wide variety of genetic and molecular manipulations, controlled manipulation of endogenous genes by, for example, gene targeting has proved elusive until relatively recently. In this review, we describe and discuss the different methods available for the inactivation and modification of endogenous loci with a focus on strategies that permit some measure of control in this process. We describe methods that use random mutagenesis to isolate mutations in specific genes. We then focus on techniques that allow controlled manipulation of the genome: gene modification by transposon mobilisation, gene knock-out mediated by zinc-finger nucleases, and gene targeting by biolistic transformation.

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AF10 (MLLT10) prevents somatic cell reprogramming through regulation of DOT1L-mediated H3K79 methylation

Epigenetics & Chromatin ◽

10.1186/s13072-021-00406-7 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Deniz Uğurlu-Çimen ◽

Deniz Odluyurt ◽

Kenan Sevinç ◽

Nazlı Ezgi Özkan-Küçük ◽

Burcu Özçimen ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cells ◽

Cell Reprogramming ◽

Cell Identity ◽

Knock Out ◽

Somatic Cell Reprogramming ◽

Proteomic Approach ◽

Reprogramming Efficiency ◽

Induced Pluripotent ◽

H3k79 Methylation

Abstract Background The histone H3 lysine 79 (H3K79) methyltransferase DOT1L is a key chromatin-based barrier to somatic cell reprogramming. However, the mechanisms by which DOT1L safeguards cell identity and somatic-specific transcriptional programs remain unknown. Results We employed a proteomic approach using proximity-based labeling to identify DOT1L-interacting proteins and investigated their effects on reprogramming. Among DOT1L interactors, suppression of AF10 (MLLT10) via RNA interference or CRISPR/Cas9, significantly increases reprogramming efficiency. In somatic cells and induced pluripotent stem cells (iPSCs) higher order H3K79 methylation is dependent on AF10 expression. In AF10 knock-out cells, re-expression wild-type AF10, but not a DOT1L binding-impaired mutant, rescues overall H3K79 methylation and reduces reprogramming efficiency. Transcriptomic analyses during reprogramming show that AF10 suppression results in downregulation of fibroblast-specific genes and accelerates the activation of pluripotency-associated genes. Conclusions Our findings establish AF10 as a novel barrier to reprogramming by regulating H3K79 methylation and thereby sheds light on the mechanism by which cell identity is maintained in somatic cells.

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Chapter 4: Pathogenesis of TBE with a focus on molecular mechanisms

Tick-borne encephalitis - The Book ◽

10.33442/26613980_4-3 ◽

2020 ◽

Keyword(s):

Immune Response ◽

Immune Responses ◽

Reverse Genetics ◽

Molecular Mechanisms ◽

Innate Immune ◽

Adaptive Immune Responses ◽

Knock Out ◽

Adaptive Immune ◽

Tick Borne Encephalitis ◽

Tick Borne Encephalitis Virus

In this chapter we describe the pathogenesis of tick-borne encephalitis virus (TBEV). To cause infection, TBEV needs to cross three different barriers; the physical, the innate and adaptive and the blood-brain barrier. The trigger of innate immune and adaptive immune responses, by TBEV is necessary to clear the infection. TBEV employs strategies to evade the innate immune response. Tools to study TBEV pathogenicity such as mouse knock-out models and reverse genetics are also discussed.

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CRISPR Turbo Accelerated Knock Out (CRISPy TAKO) for rapid in vivo screening of gene function

10.1101/2020.08.04.236968 ◽

2020 ◽

Cited By ~ 1

Author(s):

SL Plasil ◽

A Seth ◽

GE Homanics

Keyword(s):

Gene Function ◽

Reverse Genetics ◽

Behavioral Outcomes ◽

Genetic Alterations ◽

Behavioral Changes ◽

Knock Out ◽

Dna And Rna ◽

Sedative Effects ◽

Mock Treatment

AbstractThe development of CRISPR/Cas9 technology has vastly sped up the process of genome editing by introducing a bacterial system that can be exploited for reverse genetics-based research. However, generating homozygous knockout (KO) animals using traditional CRISPR/Cas9-mediated techniques requires three generations of animals. A founder animal with a desired mutation is crossed to produce heterozygous F1 offspring which are subsequently interbred to generate homozygous F2 KO animals. This study describes a novel adaptation of the CRISPR/Cas9-mediated method to develop a homozygous gene-targeted KO animal cohort in one generation. A well-characterized ethanol-responsive gene, MyD88, was chosen as a candidate gene for generation of MyD88-/- mice as proof of concept. Previous studies have reported changes in ethanol-related behavioral outcomes in MyD88 KO mice. Therefore, it was hypothesized that a successful one-generation KO of MyD88 should reproduce decreased responses to ethanols sedative effects, as well as increased ethanol consumption in males that were observed in previous studies. One-cell mouse embryos were simultaneously electroporated with four gRNAs targeting a critical Exon of MyD88 along with Cas9. DNA and RNA analysis of founder mice revealed a complex mix of genetic alterations, all of which were predicted to ablate MyD88 gene function. This study additionally compared responses of Mock treatment control mice generated through electroporation to controls purchased from a vendor. No substantial behavioral changes were noted between control cohorts. Overall, the CRISPR/Cas9 KO protocol reported here, which we call CRISPR Turbo Accelerated KnockOut (CRISPy TAKO), will be useful for reverse genetic in vivo screens of gene function in whole animals.

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