Genome Editing in Hymenoptera

Insect Biochemistry and Molecular Biology, Год журнала: 2025, Номер 180, С. 104300 - 104300

Опубликована: Март 11, 2025

The application of genome editing tools in Hymenoptera has transformative potential for functional genetics and understanding their unique biology. comprise one the most diverse Orders animals, development methods efficiently creating precise modifications could have applications conservation, pest management agriculture. To date, sex determination, DNA methylation, taste smell sensory systems as well phenotypic markers been selected gene investigations. From these data, insights into eusociality, nature haplodiploidy complex communication that possess provided an evolutionary history led them to become so successful. Insights from analyses supported by ever-improving suite CRIPSR further expansion will allow more specific biological hypotheses be tested beyond lab. Looking ahead, Hymenopteran modifying biocontrol agents agricultural pests use managing invasive species through technologies such drives. This review provides accessibility information regarding status editing, intending support considered CRISPR novel innovation refinement which it already achieved.

Язык: Английский

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Oocyte electroporation prior to in vitro fertilization is an efficient method to generate single, double, and multiple knockout porcine embryos of interest in biomedicine and animal production

Theriogenology, Год журнала: 2024, Номер 218, С. 111 - 118

Опубликована: Фев. 1, 2024

Genetically modified pigs play a critical role in mimicking human diseases, xenotransplantation, and the development of resistant to viral diseases. The use programmable endonucleases, including CRISPR/Cas9 system, has revolutionized generation genetically pigs. This study evaluates efficiency electroporation oocytes prior fertilization generating edited gene embryos for different models. For single editing, phospholipase C zeta (PLC ζ) fused sarcoma (FUS) genes were used, concentration sgRNA Cas9 complexes was optimized. results showed that increasing resulted higher mutation rates without affecting blastocyst rate. Electroporation produced double knockouts TPC1/TPC2 with high (79 %). In addition, resistance diseases such as PRRS swine influenza achieved by electroporation, allowing knockout embryo (63 also demonstrated potential multiple editing step using which is relevant xenotransplantation. technique simultaneous 5 (GGTA1, B4GALNT2, pseudo CMAH GHR). Overall, proved be an efficient versatile method generate embryonic pigs, offering significant advances biomedical agricultural research, disease resistance. led processing numerous session less expensive equipment. We confirmed gene-edited porcine single, double, or quintuple simultaneously altering stage. provide valuable insights into optimization protocols models, opening new avenues research applications this field.

Язык: Английский

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Protocol for the electroporation of CRISPR-Cas for DNA and RNA targeting in Bos taurus zygotes

STAR Protocols, Год журнала: 2024, Номер 5(1), С. 102940 - 102940

Опубликована: Март 1, 2024

The use of CRISPR-Cas9 ribonucleoproteins has revolutionized manipulation genomes. Here, we present a protocol for the electroporation CRISPR-Cas DNA and RNA targeting in Bos taurus zygotes. First, describe steps production preparation presumptive zygotes electroporation. first introduces formed by Cas9D10A with two guide RNAs to target DNA, second same ribonucleoprotein complex plus Cas13a one RNAs. For complete details on execution this protocol, please refer Nix et al.1

Язык: Английский

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Tackling mosaicism in gene edited livestock

Frontiers in Animal Science, Год журнала: 2024, Номер 5

Опубликована: Июнь 3, 2024

The farming of livestock has a critical role to play in global nutritional security and poverty alleviation. To meet these goals through more efficient, environmentally sustainable animal welfare focused means, gene editing technologies could be integrated into current breeding programs. A common issue with zygotes is the high incidence genetic mosaicism. Genetic mosaicism, characterised by single individual carrying distinct genotypes different cell lineages, can lead inconsistent presentation desired trait phenotypically, or absence intended genotype animal’s germline. This review explores present status mosaicism associated CRISPR-Cas9 cattle, sheep, pigs, identifies four areas for refinement; (1) type genome editor used; (2) formats timing during embryonic development; (3) method used deliver (4) screening strategies applied. We also discuss alternatives direct zygote editing, including surrogate sire technology blastomere separation, which circumvent production mosaic offspring. By exploring avenues reducing protocols become efficient effective, will ultimately pave way traits introduced that improve standards help address gaps nutrition access.

Язык: Английский

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Optimized Cas9:sgRNA delivery efficiently generates biallelic MSTN knockout sheep without affecting meat quality

BMC Genomics, Год журнала: 2022, Номер 23(1)

Опубликована: Май 6, 2022

Abstract Background CRISPR/Cas9-based genome-editing systems have been used to efficiently engineer livestock species with precise genetic alterations intended for biomedical and agricultural applications. Previously, we successfully generated gene-edited sheep goats via one-cell-stage embryonic microinjection of a Cas9 mRNA single-guide RNAs (sgRNAs) mixture. However, most animals produced using this approach were heterozygotes. Additionally, non-homozygous gene-editing outcomes may not fully generate the desired phenotype in an efficient manner. Results We report optimization mRNA-sgRNA delivery system homozygous myostatin ( MSTN ) knockout improved growth meat production. Firstly, sgRNA selection software (sgRNAcas9) was preliminarily screen highly sgRNAs. Ten sgRNAs targeting gene selected validated vitro fibroblast cells. Four out ten (two exon 1 two 2) showed efficiency > 50%. To determine optimal CRISPR/Cas9 concentration, four levels three mixtures injected into embryos. Microinjection 100 ng/μL 200 resulted efficiency. both -knockout approximately 50% efficiency, reaching 25%. Growth rate quality -edited lambs also investigated. exhibited increased body weight average daily gain. Moreover, pH, drip loss, intramuscular fat, crude protein, shear force gluteal muscles did show changes compared wild-type lambs. Conclusions This study highlights importance evaluation dosage editing reagents. enabled engineering sheep. confirms that does negatively impact quality, thus supporting adoption as tool improve productivity farm animals.

Язык: Английский

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Optimising Electroporation Condition for CRISPR/Cas-Mediated Knockout in Zona-Intact Buffalo Zygotes

Animals, Год журнала: 2023, Номер 14(1), С. 134 - 134

Опубликована: Дек. 30, 2023

Somatic cell nuclear transfer or cytoplasm microinjection has widely been used to produce genome-edited farm animals; however, these methods have several drawbacks which reduce their efficiency. In the present study, we describe an easy adaptable approach for introduction of mutations using CRISPR-Cas9 electroporation zygote (CRISPR-EP) in buffalo. The goal study was determine optimal conditions experimental method CRISPR/Cas9 system is introduced into vitro-produced buffalo zygotes by electroporation. Electroporation performed different combinations voltage, pulse and time, observed that at 20 V/mm, 5 pulses, 3 msec 10 h post insemination (hpi) resulted increased membrane permeability higher knockout efficiency without altering embryonic developmental potential. Using above parameters, targeted POU5F1 gene as a proof concept found no variations competence cleavage blastocyst formation rate between control, POU5F1-KO, electroporated control (EC) embryos. To elucidate effect POU5F1-KO on other pluripotent genes, determined relative expression SOX2, NANOG, GATA2 (POU5F1 intact) POU5F1-KO-confirmed blastocyst. significantly (p ≤ 0.05) altered stage conclusion, standardized straightforward protocol CRISPR-EP could be served useful studying functional genomics

Язык: Английский

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Establishment of CRISPR-Cas9 ribonucleoprotein mediated MSTN gene edited pregnancy in buffalo: Compare cells transfection and zygotes electroporation

Theriogenology, Год журнала: 2024, Номер 229, С. 158 - 168

Опубликована: Авг. 23, 2024

Язык: Английский

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Commercial perspectives: Genome editing as a breeding tool for health and well-being in dairy cattle

JDS Communications, Год журнала: 2024, Номер 5(6), С. 767 - 771

Опубликована: Сен. 3, 2024

Genome editing is the latest breeding tool capable of accelerating rate genetic improvement for health and well-being traits in food animals. It enables introduction beneficial alleles within a single generation, including those low frequency or absent population, while effectively bypassing linkage drag. For dairy industry, genome can be used to make rapid improvements that are precise, efficient, transgene-free functional not practically addressed without disrupting conventional goals overall economic merit based on genomic selection. Herein, various case studies cattle presented demonstrate applications enhancing heat stress tolerance, reduced disease susceptibility, and/or other qualitative some breeds. One highlights success simultaneous multiple loci through recent advancements embryonic stem cell biology. Multiplexed crucial addressing polygenic nature inherent many economically important livestock. However, maximizing benefits depends continued discovery targets commercially important. Commercialization also rapidly evolving regulatory statutes risk assessment, where countries already permit commercialization with non-GMO alterations existing regulations. New technologies like now poised have significant impact equipping elite performance more resilient infectious climate change loss production gains obtained from decades

Язык: Английский

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Transgenesis and Genome Engineering: A Historical Review

Methods in molecular biology, Год журнала: 2023, Номер unknown, С. 1 - 32

Опубликована: Янв. 1, 2023

Язык: Английский

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Perspectives in Genome-Editing Techniques for Livestock

Animals, Год журнала: 2023, Номер 13(16), С. 2580 - 2580

Опубликована: Авг. 10, 2023

Genome editing of farm animals has undeniable practical applications. It helps to improve production traits, enhances the economic value livestock, and increases disease resistance. Gene-modified are also used for biomedical research drug demonstrate potential be as xenograft donors humans. The recent discovery site-specific nucleases that allow precision genome a single-cell embryo (or embryonic stem cells) development new embryological delivery manipulations have revolutionized transgenesis field. These relatively approaches already proven efficient reliable engineering wide use in agriculture. A number advanced methodologies been tested laboratory models might considered application livestock animals. At same time, these methods must meet requirements safety, efficiency availability their range This review aims at covering brief history animal outlines possible future directions design optimal cost-effective tools species.

Язык: Английский

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