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Strategies to reduce genetic mosaicism following CRISPR-mediated genome edition in bovine embryos.

ABSTRACT: Genetic mosaicism is the presence of more than two alleles on an individual and it is commonly observed following CRISPR microinjection of zygotes. This phenomenon appears when DNA replication precedes CRISPR-mediated genome edition and it is undesirable because it reduces greatly the odds for direct KO generation by randomly generated indels. In this study, we have developed alternative protocols to reduce mosaicism rates following CRISPR-mediated genome edition in bovine. In a preliminary study we observed by EdU incorporation that DNA replication has already occurred at the conventional microinjection time (20?hpi). Aiming to reduce mosaicism appearance, we have developed three alternative microinjection protocols: early zygote microinjection (10?hpi RNA) or oocyte microinjection before fertilization with either RNA or Ribonucleoprotein delivery (0 hpi RNA or 0 hpi RNP). All three alternative microinjection protocols resulted in similar blastocyst and genome edition rates compared to the conventional 20 hpi group, whereas mosaicism rates were significantly reduced in all early delivery groups (~10-30% of edited embryos being mosaic depending on the loci) compared to conventional 20 hpi microinjection (100% mosaicism rate). These strategies constitute an efficient way to reduce the number of indels, increasing the odds for direct KO generation.

SUBMITTER: Lamas-Toranzo I

PROVIDER: S-EPMC6797768 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Strategies to reduce genetic mosaicism following CRISPR-mediated genome edition in bovine embryos.

Lamas-Toranzo I I Galiano-Cogolludo B B Cornudella-Ardiaca F F Cobos-Figueroa J J Ousinde O O Bermejo-Álvarez P P

Scientific reports 20191017 1

Genetic mosaicism is the presence of more than two alleles on an individual and it is commonly observed following CRISPR microinjection of zygotes. This phenomenon appears when DNA replication precedes CRISPR-mediated genome edition and it is undesirable because it reduces greatly the odds for direct KO generation by randomly generated indels. In this study, we have developed alternative protocols to reduce mosaicism rates following CRISPR-mediated genome edition in bovine. In a preliminary stud ...[more]

PMID: 31624292

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Project description:The mitochondrial transcription factor A (TFAM) is a mitochondrial DNA (mtDNA) binding protein essential for the initiation of transcription and genome maintenance. Recently it was demonstrated that the primary role of TFAM is to maintain the integrity of mtDNA and that it is a key regulator of mtDNA copy number. It was also shown that TFAM plays a central role in the mtDNA stress-mediated inflammatory response. In our study, we proposed to evaluate the possibility of editing the TFAM gene by CRISPR/Cas9 technology in bovine fibroblasts, as TFAM regulates the replication specificity of mtDNA. We further attempted to maintain these cells in culture post edition in a medium supplemented with uridine and pyruvate to mimic Rho zero cells that are capable of surviving without mtDNA, because it is known that the TFAM gene is lethal in knockout mice and chicken. Moreover, we evaluated the effects of TFAM modification on mtDNA copy number. The CRISPR gRNA was designed to target exon 1 of the bovine TFAM gene and subsequently cloned. Fibroblasts were transfected with Cas9 and control plasmids. After 24 h of transfection, cells were analyzed by flow cytometry to evaluate the efficiency of transfection. The site directed-mutation frequency was assessed by T7 endonuclease assay, and cell clones were analyzed for mtDNA copy number by Sanger DNA sequencing. We achieved transfection efficiency of 51.3%. We selected 23 successfully transformed clones for further analysis, and seven of these exhibited directed mutations at the CRISPR/Cas9 targeted site. Moreover, we also found a decrease in mtDNA copy number in the gene edited clones compared to that in the controls. These TFAM gene mutant cells were viable in culture when supplemented with uridine and pyruvate. We conclude that this CRISPR/Cas9 design was efficient, resulting in seven heterozygous mutant clones and opening up the possibility to use these mutant cell lines as a model system to elucidate the role of TFAM in the maintenance of mtDNA integrity.

Project description:BackgroundGenetic mosaicism is commonly observed in human blastocysts. Embryos' morphokinetic feature observed from time-lapse monitoring (TLM) is helpful to predict the embryos' ploidy status in a non-invasive way. However, morphokinetic research on mosaic embryos is extremely limited. Moreover, transfer of mosaic embryos is a new attempt in reproductive medicine, while studies regarding the clinical and neonatal outcomes following transfer of embryos with different levels and types of mosaicism are needed. This study aimed to investigate the morphokinetic characteristics of mosaic blastocysts, uncover clinical outcomes of mosaic embryos, and evaluate the effect of level and type of mosaicism on transfer outcomes.ResultsA total of 923 blastocysts from 229 preimplantation genetic testing cycles were cultured in TLM incubators in a single fertilization center between July 2016 and July 2021. Multivariate logistic regression models showed mosaic embryos had significantly shorter time to reach morula when compared with euploid (P = 0.002), mosaic with aneuploid (P = 0.005), and aneuploid (P = 0.005) embryos after adjusting the potential confounders. KIDScore is an artificial intelligence scoring program from time lapse incubation system to predict embryo implantation potential. Mosaic with aneuploid embryos had significantly lower KIDScore than euploid (P = 6.47e-4), mosaic (P = 0.005), and aneuploid (P = 0.004) embryos after adjustment. Meanwhile, we compared the clinical outcomes following transfer of low-level (< 50%) mosaic embryos (N = 60) with euploid embryos (N = 1301) matched using propensity scoring collected from September 2020 to January 2023. Mosaic embryos had significantly lower clinical pregnancy rate (41.67% vs. 57.65%, P = 0.015) and live birth rate (38.33% vs. 51.35%, P = 0.048) than the euploid embryos. Subgroup analyses showed the whole, segmental, and complex chromosome mosaic embryos had the similar clinical outcomes.ConclusionsThe shortened time to reach morula in mosaic embryos and the low KIDScore in mosaic with aneuploid embryos revealed innovative clues to embryo selection with the non-invasive TLM and provided new insights into biological mechanism of chromosomal abnormality. The analyses of overall and subgroups of mosaic embryo transfer outcomes helped to optimize embryo transfer scheme for in-vitro fertilization procedures. Multi-center prospective studies with large sample sizes are warranted to validate our results in the future.

Project description:BACKGROUND: Bovine Herpesvirus type-5 (BoHV-5) is a neurovirulent ?-Herpesvirus which is potentially pathogenic for cows and suspected to be associated with reproductive disorders. Interestingly, natural transmission of BoHV-5 by contaminated semen was recently described in Australia. Additionally, BoHV-5 was also isolated from the semen of a healthy bull in the same country and incriminated in a natural outbreak of reproductive disease after artificial insemination. In contrast with BoHV-1, experimental exposure of in vitro produced bovine embryos to BoHV-5 does not affect embryo viability and seems to inhibit some pathways of apoptosis. However, the mechanisms responsible for these phenomena are poorly understood. In this study, we examined mitochondrial activity, antioxidant protection, stress response and developmental rates of in vitro produced bovine embryos that were exposed and unexposed to BoHV-5. METHODS: For this purpose, bovine embryos produced in vitro were assayed for cell markers after experimental infection of oocytes (n?=?30; five repetitions), in vitro fertilization and development. The indirect immunofluorescence was employed to measure the expression of superoxide dismutase 1 (SOD1), anti-oxidant like protein 1 (AOP-1), heat shock protein 70.1 (Hsp 70.1) and also viral antigens in embryos derived from BoHV-5 exposed and unexposed oocytes. The determination of gene transcripts of mitochondrial activity (SOD1), antioxidant protection (AOP-1) and stress response (Hsp70.1) were evaluated using the reverse transcriptase polymerase chain reaction (RT-PCR). MitoTracker Green FM, JC-1 and Hoechst 33342-staining were used to evaluate mitochondrial distribution, segregation patterns and embryos morphology. The intensity of labeling was graded semi-quantitatively and embryos considered intensively marked were used for statistical analysis. RESULTS: The quality of the produced embryos was not affected by exposure to BoHV-5. Of the 357 collected oocytes, 313 (+/- 6.5; 87.7%) were cleaved and 195 (+/- 3.2; 54.6%) blastocysts were produced without virus exposure. After exposure, 388 oocytes were cleaved into 328 (+/- 8.9, 84.5%), and these embryos produced 193 (+/- 3.2, 49.7%) blastocysts. Viral DNA corresponding to the US9 gene was only detected in embryos at day 7 after in vitro culture, and confirmed by indirect immunofluorescence assay (IFA). These results revealed significant differences (p?<?0.05) between exposed and unexposed oocytes fertilized, as MitoTracker Green FM staining Fluorescence intensity of Jc-1 staining was significantly higher (p?<?0.005) among exposed embryos (143 +/- 8.2). There was no significant difference between the ratios of Hoechst 33342-stained nuclei and total cells in good-quality blastocysts (in both the exposed and unexposed groups). Using IFA and reverse transcriptase polymerase chain reaction (RT-PCR) for the set of target transcripts (SOD1, AOP-1 and Hsp 70.1), there were differences in the mRNA and respective proteins between the control and exposed embryos. Only the exposed embryos produced anti-oxidant protein-like 1 (AOP-1). However, neither the control nor the exposed embryos produced the heat shock protein Hsp 70.1. Interestingly, both the control and the exposed embryos produced superoxide dismutase (SOD1), revealing intense mitochondrial activity. CONCLUSION: This is the first demonstration of SOD1 and AOP-1 production in bovine embryos exposed to BoHV-5. Intense mitochondrial activity was also observed during infection, and this occurred without interfering with the quality or number of produced embryos. These findings further our understanding on the ability of ?-Herpesviruses to prevent apoptosis by modulating mitochondrial pathways.

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Strategies to reduce genetic mosaicism following CRISPR-mediated genome edition in bovine embryos.

Publications

Strategies to reduce genetic mosaicism following CRISPR-mediated genome edition in bovine embryos.

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