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

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.

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Precise genome engineering inDrosophilausing prime editing

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2021996118 ◽

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.

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What can PIWI-interacting RNA research learn from chickens, and vice versa?

Canadian Journal of Animal Science ◽

10.1139/cjas-2018-0252 ◽

2019 ◽

Vol 99 (4) ◽

pp. 641-648

Author(s):

Xin Zhiguo Li

Keyword(s):

Germ Line ◽

Rapid Evolution ◽

Fruit Fly ◽

Model System ◽

Arms Race ◽

P Element ◽

Model Organisms ◽

Reproductive Efficiency ◽

Common Features ◽

Adaptive Immune

P-element induced wimpy testis (PIWI) interacting RNA (piRNA) are essential for fertility, by protecting the integrity of the germ-line genome via silencing of transposable elements (TE). Because new TE are constantly invading the host genome, piRNA-producing loci are under continuous pressure to undergo rapid evolution. This arms race between TE and piRNA is a prime example of the genome being more plastic than previously thought. Historically, the study of piRNA and TE has benefited from the use of diverse model organisms, including worms, fruit fly, zebrafish, frogs, and mice. In domestic chickens, we recently identified a new mode of piRNA acquisition in which the host hijacks and converts a pre-existing provirus into a piRNA-producing locus to defend against Avian leukosis virus, an adaptive immune strategy similar to the prokaryotic CRISPR–Cas [clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas)] system. This finding reveals a previously unrecognized mechanism of the host piRNA repertoire to rapidly evolve and target TE specifically. In this review, we will focus on both the unique and common features of chicken piRNA, as well as the advantages of using chickens as a model system, to address fundamental questions regarding piRNA acquisition in hosts. We will also comment on the potential application of piRNA for improving poultry health and reproductive efficiency.

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Precise genome engineering in Drosophila using prime editing

10.1101/2020.08.05.232348 ◽

2020 ◽

Cited By ~ 3

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.

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Epigenetic Events and Germ Line Transmission in Rag1 knock-out Chimeric Mice Produced by CRISPR/Cas9 Technology

10.21203/rs.3.rs-153876/v1 ◽

2021 ◽

Author(s):

Maryam Salimi ◽

Maryam Mehravar ◽

Abolfazl Shirazi ◽

Ali Sarvari ◽

Mohammad Mahdi Naderi ◽

...

Keyword(s):

Embryonic Development ◽

Germ Line ◽

Methylation Status ◽

Immunohistochemical Analysis ◽

Normal Female ◽

Epigenetic Alterations ◽

Chimeric Mice ◽

Knock Out ◽

Transgenic Embryos ◽

Germ Line Transmission

Abstract Considering the effects of epigenetic changes on the embryonic development through altering the gene expression of pluripotency, trophectoderm, and imprinting genes, we determined the pattern of epigenetic alterations in transgenic embryos produced by injecting transgenic mESCs into the morula. To aim this, RAG1 knock-out mESCs were produced using CRISPR-Cas9 editing system and then microinjected into morulas to develop chimeric embryos. Afterward, the expression of pluripotency, trophectoderm genes, and imprinting genes in these embryos were evaluated using real-time PCR. Immunohistochemical analysis was carried out to determine the methylation rates of H3K9me3 and H3K4me3. In the following, since epigenetic alterations can be at least one of the possible reasons of male infertility and the loss of germ-line transmission capacity, the germ-line transmission ability in chimeric mice was also evaluated by breeding them and subsequent backcrosses to wild type strains. Our findings showed that the methylation rates of H3K4me3 and H3K9me3 were significantly lower and higher respectively in the RAG1 knock-out embryos compared others groups. Moreover, the chimeric embryos exhibited the decreased expression of NANOG, OCT4, CDX2, TEAD4, and H19 genes. Following the breeding of chimeric males with normal female mouse, 40% of chimeras had no germ-line transmission (GLT), and 60% were infertile.We showed that the manipulation of mESC by CRISPR-Cas9 approach remarkably changed the methylation status of H3K9me3 and H3K4me3, resulting in impaired development of embryos through dysregulation of genes involved embryonic development and then, may be one of the reasons of infertility and lack of GLT.

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BREC: an R package/Shiny app for automatically identifying heterochromatin boundaries and estimating local recombination rates along chromosomes

BMC Bioinformatics ◽

10.1186/s12859-021-04233-1 ◽

2021 ◽

Vol 22 (S6) ◽

Author(s):

Yasmine Mansour ◽

Annie Chateau ◽

Anna-Sophie Fiston-Lavier

Keyword(s):

Data Quality ◽

Data Science ◽

Fruit Fly ◽

R Package ◽

Model Organisms ◽

Data Quality Control ◽

Recombination Rates ◽

Functional Dynamics ◽

Shiny App ◽

User Friendly

Abstract Background Meiotic recombination is a vital biological process playing an essential role in genome's structural and functional dynamics. Genomes exhibit highly various recombination profiles along chromosomes associated with several chromatin states. However, eu-heterochromatin boundaries are not available nor easily provided for non-model organisms, especially for newly sequenced ones. Hence, we miss accurate local recombination rates necessary to address evolutionary questions. Results Here, we propose an automated computational tool, based on the Marey maps method, allowing to identify heterochromatin boundaries along chromosomes and estimating local recombination rates. Our method, called BREC (heterochromatin Boundaries and RECombination rate estimates) is non-genome-specific, running even on non-model genomes as long as genetic and physical maps are available. BREC is based on pure statistics and is data-driven, implying that good input data quality remains a strong requirement. Therefore, a data pre-processing module (data quality control and cleaning) is provided. Experiments show that BREC handles different markers' density and distribution issues. Conclusions BREC's heterochromatin boundaries have been validated with cytological equivalents experimentally generated on the fruit fly Drosophila melanogaster genome, for which BREC returns congruent corresponding values. Also, BREC's recombination rates have been compared with previously reported estimates. Based on the promising results, we believe our tool has the potential to help bring data science into the service of genome biology and evolution. We introduce BREC within an R-package and a Shiny web-based user-friendly application yielding a fast, easy-to-use, and broadly accessible resource. The BREC R-package is available at the GitHub repository https://github.com/GenomeStructureOrganization.

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Direct Estimate of the Mutation Rate and the Distribution of Fitness Effects in the Yeast Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/159.2.441 ◽

2001 ◽

Vol 159 (2) ◽

pp. 441-452

Author(s):

Dominika M Wloch ◽

Krzysztof Szafraniec ◽

Rhona H Borts ◽

Ryszard Korona

Keyword(s):

Spontaneous Mutation ◽

Fruit Fly ◽

Diploid Cell ◽

Induced Mutations ◽

Direct Estimate ◽

Yeast Saccharomyces Cerevisiae ◽

Related Trait ◽

First Time ◽

Defective Mismatch Repair

Abstract Estimates of the rate and frequency distribution of deleterious effects were obtained for the first time by direct scoring and characterization of individual mutations. This was achieved by applying tetrad analysis to a large number of yeast clones. The genomic rate of spontaneous mutation deleterious to a basic fitness-related trait, that of growth rate, was U = 1.1 × 10−3 per diploid cell division. Extrapolated to the fruit fly and humans, the per generation rate would be 0.074 and 0.92, respectively. This is likely to be an underestimate because single mutations with selection coefficients s < 0.01 could not be detected. The distribution of s ≥ 0.01 was studied both for spontaneous and induced mutations. The latter were induced by ethyl methanesulfonate (EMS) or resulted from defective mismatch repair. Lethal changes accounted for ~30–40% of the scored mutations. The mean s of nonlethal mutations was fairly high, but most frequently its value was between 0.01 and 0.05. Although the rate and distribution of very small effects could not be determined, the joint share of such mutations in decreasing average fitness was probably no larger than ~1%.

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Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6755-6758.1990 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6755-6758

Author(s):

B R Stanton ◽

S W Reid ◽

L F Parada

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Homologous Recombination ◽

Embryonic Development ◽

Cell Lines ◽

Germ Line ◽

Es Cells ◽

Embryonic Stem ◽

Es Cell ◽

Germ Line Transmission

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.

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Distribution, expression and germ line transmission of exogenous DNA sequences following microinjection into Xenopus laevis eggs

Development ◽

10.1242/dev.99.1.15 ◽

1987 ◽

Vol 99 (1) ◽

pp. 15-23

Author(s):

L.D. Etkin ◽

B. Pearman

Keyword(s):

Xenopus Laevis ◽

Dna Sequences ◽

Copy Number ◽

Germ Line ◽

Gene Promoter ◽

Early Gene ◽

Mosaic Pattern ◽

Exogenous Dna ◽

Gene Coding ◽

Germ Line Transmission

We analysed the fate, expression and germ line transmission of exogenous DNA which was microinjected into fertilized eggs of Xenopus laevis. DNA was injected into fertilized eggs within 1 h following fertilization. The injected DNA was dispersed around the site of injection and became localized to cleavage nuclei by stage 6. Injected DNA persisted in the tissues of 6- to 8-month-old frogs and exhibited a mosaic pattern of distribution with regard to the presence or absence and copy number between different tissues. We detected the exogenous DNA sequences in 60% of injected frogs. Restriction digestion analysis of this DNA suggested that it is not rearranged and was organized as head-to-tail multimers. The copy number varied from 2 to 30 copies/cell in various tissues of the same frog. Plasmid pSV2CAT which contains the prokaryotic gene coding for chloramphenicol acetyl transferase (CAT) enzyme linked to the SV40 early gene promoter was expressed in 50% of the animals containing the gene. The pattern of expression, however, varied between different animals and could not be correlated with copy number. We also showed that the exogenous DNA sequences were transmitted through the male germ line and that each offspring contained the gene integrated into a different region of the genome.

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