CRISPR/Cas9-mediated base editors and their prospects for mitochondrial genome engineering.

Base editors are a type of double-stranded break (DSB)-free gene editing technology that has opened up new possibilities for precise manipulation of mitochondrial DNA (mtDNA). This includes cytosine and adenosine base editors and more recently guanosine base editors. Because of having low off-target and indel rates, there is a growing interest in developing and evolving this research field. Here, we provide a detailed update on DNA base editors. While base editing has widely been used for nuclear genome engineering, the growing interest in applying this technology to mitochondrial DNA has been faced with several challenges. While Cas9 protein has been shown to enter mitochondria, use of smaller Cas proteins, such as Cas12a, has higher import efficiency. However, sgRNA transfer into mitochondria is the most challenging step. sgRNA structure and ratio of Cas protein to sgRNA are both important factors for efficient sgRNA entry into mitochondria. In conclusion, while there are still several challenges to be addressed, ongoing research in this field holds the potential for new treatments and therapies for mitochondrial disorders.

An Electrochemical Protocol for CRISPR-Mediated Gene-Editing of Sheep Embryonic Fibroblast Cells.

Genetic engineering of farm animals is commonly carried out via cell-mediated transfection followed by somatic cell nuclear transfer. However, efficient transfer of exogenous DNA into ovine embryonic fibroblast (EF) cells without compromising cell viability has remained a challenging issue. Here, we aimed to develop a protocol for electrotransfection of sheep EF cells. First, we optimized the pulsing condition using an OptiMEM-GlutaMAX medium as the electroporation buffer and found 2 pulses of 270 V, each for 10 ms and 10 s interval, is the most efficient condition to have a high rate of transfection and cell survival. Moreover, supplementing 3% dimethyl sulfoxide (DMSO) into the electroporation medium considerably improved the cell viability after the electroporation process. The electroporation procedure resulted in >98% transfection efficiency and >97% cell survival rate using reporter plasmids. Finally, using CRISPR/Cas9-encoding vectors, we targeted BMP15 and GDF9 genes in sheep EF cells. The electroporated cells are associated with a 52% indels rate using single gRNAs as well as a highly efficient target deletion using 2 gRNAs. In conclusion, we have developed an electrotransfection protocol using the OptiMEM-GlutaMAX medium supplemented with 3% DMSO for sheep EF cells. The electroporation method can be used for cell-mediated gene-editing in sheep.

Cotreatment of IGF1 and Fadrozole Upregulates the Expression of RSPO1, SOX9, and AMH in Chicken Embryos.

Insulin-like growth factor-1 (IGF1) and anti-aromatase synergistically increase the rate and stability of female-to-male sex reversal as well as pre- and postnatal weight gains in hatched chickens. This study aimed at assessing gene expression profiles of chicken embryos treated with IGF1 and fadrozole. Day 3.5 fertile eggs were in ovo injected with one of IGF1, fadrozole anti-aromatase, combined IGF1 and fadrozole, or sham injection. The expression profile was studied on day 6 and day 11 of the embryonic development following gonadal differentiation. On day 6 of embryonic development, simultaneous injection of IGF1 and fadrozole significantly upregulated the expression of RSPO1, AMH, and SOX9 in genetically female embryos compared to single injections and control groups. Also, a higher expression of ESR1 and BMP4 was observed in genetically male embryos on day 6 compared to the control group. In day 11 embryos, a higher expression of BMP4 was detected in both males and females of the IGF1 and fadrozole-administered group compared to the sham injection cohort. In conclusion, the results of this study indicate that combined effects of IGF1 and fadrozole induce female-to-male sex reversal by increasing the expression of testis developmental factors rather than attenuating ovary developmental factors.

Is passive transmission of non-viral vectors through artificial insemination of sperm-DNA mixtures sufficient for chicken transgenesis?

DNA uptake in the post-acrosomal region of the spermatozoa takes place exclusively in immotile spermatozoa that are naturally unable to fertilize eggs. The present study aimed to assess whether passive transmission of non-viral vectors to the surrounding areas of chicken embryos could be an alternate mechanism in chicken sperm-mediated gene transfer. First, the presence of nucleases in rooster seminal plasma was evaluated. Semen ejaculates from five roosters were centrifuged and the supernatant was incubated with pBL2 for 1 h. A robust nuclease cocktail was detected in the rooster semen. To overcome these nucleases, plasmid-TransIT combinations were incubated with semen for 1 h. Incubation of exogenous DNA in the lipoplex structure could considerably bypass the semen nuclease effect. Then, intravaginal insemination of 1 × 10(9) sperm mixed with lipoplexes (40 µg pBL2:40 µl TransIT) was carried out in 15 virgin hens. Neither the epithelial tissue from the inseminated female reproductive tracts nor the produced embryos following artificial insemination showed the transgene. To remove any bias in the transgene transmission possibility, the plasmid-TransIT admixture was directly injected in close vicinity of the embryos in newly laid eggs. Nonetheless, none of the produced fetuses or chicks carried the transgene. In conclusion, the results of the present study revealed a nuclease admixture in rooster seminal plasma, and passive/active transmission of the non-viral vector into close vicinity of the chicken embryo was inefficient for producing transgenic chicks.

Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos.

Transgenic mammals have been produced using sperm as vectors for exogenous DNA (sperm-mediated gene transfer (SMGT)) in combination with artificial insemination. Our study evaluated whether SMGT could also be achieved in combination with IVF to efficiently produce transgenic bovine embryos. We assessed binding and uptake of fluorescently labelled plasmids into sperm in the presence of different concentrations of dimethyl sulphoxide or lipofectamine. Live motile sperm displayed a characteristic punctuate fluorescence pattern across their entire surface, while uniform postacrosomal fluorescence was only apparent in dead sperm. Association with sperm or lipofection reagent protected exogenous DNA from DNase I digestion. Following IVF, presence and expression of episomal and non-episomal green fluorescent protein (GFP)-reporter plasmids was monitored in oocytes and embryos. We found no evidence of intracellular plasmid uptake and none of the resulting zygotes (n=96) and blastocysts were GFP positive by fluorescence microscopy or genomic PCR (n=751). When individual zona-free oocytes were matured, fertilised and continuously cultured in the presence of episomal reporter plasmids until the blastocyst stage, most embryos (38/68=56%) were associated with the exogenous DNA. Using anti-GFP immunocytochemistry (n=48) or GFP fluorescence (n=94), no GFP expression was detected in blastocysts. By contrast, ICSI resulted in 18% of embryos expressing the GFP reporter. In summary, exposure to DNA was an inefficient technique to produce transgenic bovine sperm or blastocysts in vitro.

CRISPR/Cas9-mediated targeted knock-in of large constructs using nocodazole and RNase HII.

On-target integration of large cassettes via homology-directed repair (HDR) has several applications. However, the HDR-mediated targeted knock-in suffered from low efficiency. In this study, we made several large plasmids (12.1-13.4 kb) which included the CRISPR/Cas9 system along with a puromycin transgene as part of the large DNA donor (5.3-7.1 kb insertion cassettes) and used them to evaluate their targeted integration efficiency into a transgenic murine embryonic fibroblast (MEF) cell line carrying a single copy of a Venus transgene. We established a detection assay by which HDR events could be discriminated from the error-prone non-homologous end-joining (NHEJ) events. Improving the plasmid quality could considerably leverage the cell toxicity impediment of large plasmids. The use of the TILD (targeted integration with linearized dsDNA) cassettes did not improve the HDR rate compared to the circular plasmids. However, the direct inclusion of nocodazole into the electroporation solution significantly improved the HDR rate. Also, simultaneous delivery of RNase HII and the donor plasmids into the electroporated cells considerably improved the HDR events. In conclusion, the results of this study showed that using cell synchronization reagents in the electroporation medium can efficiently induce HDR rate in the mammalian genome.

The effect of multiplex-PCR-assessed major pathogens causing subclinical mastitis on somatic cell profiles.

The major pathogens causing mastitis were evaluated by multiplex-polymerase chain reaction (M-PCR) with self-designed primers in four quarters of the first, third, and fifth parities in industrial, semi-industrial, and traditional dairy cattle farms in Iran. With the incidence of infection in the quarters by Staphylococcus aureus and Streptococcus agalactiae, the mean log somatic cell count (log SCC) increased from 5.06 to 5.77. The smallest changes occurred with Escherichia coli. Contagious pathogens, when compared with environmental pathogens, were more prevalent and common and created more profound quantitative and qualitative changes in SCC profiles. The second part of the study surveyed the diversity of contaminating pathogens and their effect on quantitative and qualitative profiles of somatic cells. M-PCR was used to determine the absence (M-PCR(-)) and presence of one (M-PCR(+1)), two (M-PCR(+2)), and three (M-PCR(+3)) major pathogens in raw milk samples. Quarter log SCC increased from 5.06 (for M-PCR(-1)) to 5.5 (for M-PCR(+1)), 5.7 (for M-PCR(+2)), and 6 (for M-PCR(+3)). Percent changes in polymorphonuclears (PMNs) were not significant between different quarters and parities but were significant between different farms in terms of pathogen diversity (P < 0.05). Therefore, by increasing the number of types of major pathogens involved in subclinical mastitis, SCC of udder quarters and the proportion of PMNs significantly increased, whereas the proportion of lymphocytes significantly decreased. This subject is very important in increasing the shelf life of dairy products, because PMNs are introduced to the enzymatic pools.

Supplementation of Estradiol Into Semen Extender Improved Goat Sperm Cryopreservation.

Estradiol is a steroid hormone excreted from the female gonads, mainly during the pre-estrus. However, the potential effects of estradiol are yet to be explored on sperm parameters through cryopreservation. In this study, we supplemented estradiol, 3 and 5 µM, in the goat semen extender and assessed the sperm parameters after a freeze-thawing process. Sperm motility was assessed using the computer-assisted sperm analysis system. Sperm viability and membrane integrity improved using both 3 and 5 µM concentrations of estradiol. The highest rate of progressive motility was observed in the 3 µM estradiol group. However, a higher concentration of estradiol (5 µM) reduced the progressive motility. Then, we were interested to see if the supportive effect of estradiol on sperm motility is mediated through the intracellular concentration of calcium ionophore. We supplemented the semen extender with 1 and 10 mM ethylenediaminetetraacetic acid (EDTA) and showed that 1 mM has no adverse effect on progressive sperm motility. Then, estradiol (3 µM) was supplemented with or without EDTA (1 mM) into the semen extender. Individual EDTA treatment improved the progressive sperm motility compared to the control group. However, in the presence of estradiol, EDTA treatment reduced the progressive motility compared to the individual estradiol group. This indicated a considerable interaction between estradiol and EDTA for progressive sperm motility. Indeed, EDTA reduced the supportive effects of estradiol on sperm cryopreservation parameters. These results indicated that induction of higher progressive sperm motility in response to estradiol is a calcium-dependent process, as the EDTA did completely abrogate the estradiol-mediated effect.

Variation and frequency of supernumerary teats, litter size, histological features and the fibroblast growth factor 2 (FGF-2) gene expression pattern in goats.

Historically, female domestic goats carrying multiple kids are mostly unable to express sufficient nursing ability due to a limited number of functional teats. Therefore, the functional teat is an important component in prolific goat breeding, and plays a key role in the future health of their kids. With this motivation, we wanted to investigate the phenotypic features, litter size, histology of adult female mammary glands, and the gene expression profile of the fibroblast growth factor 2 (FGF-2) gene in goats. To illustrate this, the initial dataset of the current study consists of an electronic questionnaire that includes 697 individuals (548 does and 149 bucks) of five endemic and three exotic goats from 2015 to 2020 in different geographic areas of Iran, from 59 Markhoz (MARG), 50 Azari (AZAR), 73 Busheri (BUSH), 69 Sarbisheh (SARB), 165 Mahabadi (MOHA) indigenous goats and also exotic breeds, including 183 Saanen (SANN), 39 Alpine (ALPN), and 59 Boer (BORE) goats. The results of this study confirmed that MOHA goats (4.16%), BORE (4.43%) and SANN goat breeds (5.75%) have larger litter sizes. Interestingly, the evidence gathering when SNTs occurred showed that both the BUSH and BORE goat breeds had the highest frequency of SNTs. Moreover, under the same physiological and lactation conditions, there was no statistically significant difference in histological features between the three compared does class consist of the two teats, SNTs, and four functional teats. In addition, the results of the gene expression profile significantly highlight the FGF-2 gene pattern in two teat groups compared to other SNT groups (P < 0.01). In summary, this scenario can be used to generate further research and facts on responsible candidate genes, the variations in teat numbers in goats, examining both the incidence of SNT and litter size.

Estimation of genetic parameters for 13 female fertility indices in Holstein dairy cows.

This study was aimed to assess genetic parameters for 13 traits in heifers and first-parity Holstein dairy cows. Data consisted of calving and insemination dates of 14,707 Holstein dairy cows in Isfahan province of Iran. Reproductive traits included age at first service (AFS), first service to conception (FSTC), gestation length (GL), age at first calving (AFC), calving to first service (CTFS), days open (DO), calving interval (CI), number of services per conception (NS), and non-return rate at 56 days (NRR). Model equations were optimized using GLM procedure in SAS package following genetic analysis using animal models in ASREML software. Minimum and maximum departure from normal distribution for phenotypic records belonged to AFS, NRR, GL, DO, CI and AFC, NS, FSTC, CTFS, respectively. Estimated heritability varied from 0.002 (NRR) to 0.184 (GL) in heifers and from 0.003 (NRR) to 0.153 (GL) in first-parity cows. AFS, CTFS, and GL were noticeably heritable compared to other assessed traits. Estimated absolute additive genetic correlations were in the range of 0.01 (NRR and AFS) and 0.99 (NRR and NS) in heifers and 0.07 (GL and CI) to 1 (FSTC and CI) in cows. Additive genetic correlations were antagonistic between AFS and other traits, except AFC. Interestingly, NRR which has been included in sire catalogs had the highest average absolute genetic associations with other traits.

Ultrasound monitoring of pregnancy in mini-lop rabbits.

Ultrasound plays a considerable role in human and animal reproduction in terms of early detection of pregnancy, prediction of parturition time, and diagnosis of fetal abnormalities. The present study aimed to evaluate the ultrasound implementation for monitoring of gestation in mini-lop rabbits. Fifteen heads of pubertal does were selected and kept in normal conditions of feeding and temperature. Animals were mated with three bucks from the same breed. The pregnancy monitoring was begun from five days post-mating (dpm) to kindling using a 12.5 MHz ultrasonic transducer. The examinations were performed at fixed dpm for all does (5, 7, 12, 16, 20, and 26). Furthermore, randomly selected does (2-3 does per day; one doe was fixed) were subjected to daily ultrasound examination to estimate the relationship between the ultrasound biometrics with the gestational age (GA) and days to parturition. The pregnancy rate was 80%, and the mean number of live kits at birth was 4.2 in the present study. Based on the ultrasound records, the gestation length can be divided into three tertiles of pregnancy (TOP) in rabbits. The first TOP (0-10 dpm) was monitored by detecting and measuring the gestational sac diameter from 6 to 10 dpm. The 2nd TOP (11-12 dpm) was characterized by detection and measurement of Crown Rump Length and Fetal Heart Rate. From 15 to 20 dpm, bi-parietal diameter and head circumference were positively correlated with the GA (p-value < 0.05). Abdominal circumference and femur length were detectable and measurable during the 3rd TOP (21 dpm-kindling). Pregnancy was detected as early as six dpm with acceptable markers in mini-lop rabbits. Highly significant negative correlations were detected between days to parturition and the sonographic biometrics. Three abnormal fetuses were successfully detected and described, too.

Development of a transposon-based technology for transfection of day 0 chicken embryos.

Although the chicken embryo has been a classical model for developmental studies, the lack of straightforward technologies for chicken transgenesis limited the usefulness of this animal model. Here, we assessed electroporation and lipofection approaches for in ovo transfection of Sleeping Beauty transposon system in stage X-XII chicken embryos. Electroporation of chicken embryos could transfect the trophectodermal cells. Then, a mixture of transposon lipoplexes and high concentrated carboxymethylcellulose (HCC) solution was injected into the subgerminal cavity of day 0 embryos. The lipoplex-HCC mixture substantially increased the number of trophectodermal cells expressing the reporter. Importantly, the fluorescent reporter was detected in cells inside of the embryos as well as circulation cells in the bloodstream during days 3-4 of incubation. This study provided evidence for direct in ovo transfection of early chicken embryos, though the long-term outcome of this approach warrants further studies.

A versatile bulk electrotransfection protocol for murine embryonic fibroblasts and iPS cells.

Although electroporation has been widely accepted as the main gene transfer tool, there is still considerable scope to improve the electroporation efficiency of exogenous DNAs into primary cells. Here, we developed a square-wave pulsing protocol using OptiMEM-GlutaMAX for highly efficient transfection of murine embryonic fibroblasts (MEF) and induced pluripotency stem (iPS) cells using reporter genes as well as gRNA/Cas9-encoding plasmids. An electrotransfection efficiency of > 95% was achieved for both MEF and iPS cells using reporter-encoding plasmids. The protocol was efficient for plasmid sizes ranging from 6.2 to 13.5 kb. Inducing the error prone non-homologous end joining repair by gRNA/Cas9 plasmid transfection, a high rate of targeted gene knockouts of up to 98% was produced in transgenic cells carrying a single-copy of Venus reporter. Targeted deletions in the Venus transgene were efficiently (up to 67% deletion rate) performed by co-electroporation of two gRNA-encoding plasmids. We introduced a plasmid electrotransfection protocol which is straight-forward, cost-effective, and efficient for CRISPRing murine primary cells. This protocol is promising to make targeted genetic engineering using the CRISPR/Cas9 plasmid system.

Systematic optimization of square-wave electroporation conditions for bovine primary fibroblasts.

BACKGROUND: Gene transfer by electroporation is an established method for the non-viral mediated transfection of mammalian cells. Primary cells pose a particular challenge for electroporation-mediated gene transfer, since they are more vulnerable than immortalized cells, and have a limited proliferative capacity. Improving the gene transfer by using square wave electroporation in difficult to transfect cells, like bovine fetal fibroblasts, is a prerequisite for transgenic and further downstream experiments. RESULTS: Here, bovine fetal fibroblasts were used for square-wave electroporation experiments in which the following parameters were systematically tested: electroporation buffer, electroporation temperature, pulse voltage, pulse duration, pulse number, cuvette type and plasmid DNA amount. For the experiments a commercially available square-wave generator was applied. Post electroporation, the bovine fetal fibroblasts were observed after 24 h for viability and reporter expression. The best results were obtained with a single 10 millisecond square-wave pulse of 400 V using 10 µg supercoiled plasmid DNA and 0.3 × 106 cells in 100 µl of Opti-MEM medium in 4 mm cuvettes. Importantly, the electroporation at room temperature was considerably better than with pre-cooled conditions. CONCLUSIONS: The optimized electroporation conditions will be relevant for gene transfer experiments in bovine fetal fibroblasts to obtain genetically engineered donor cells for somatic cell nuclear transfer and for reprogramming experiments in this species.

Robust DNase activity of the ooplasm can act as a gametic transfection barrier in rainbow trout.

In this study, we evaluated DNase activity of rainbow trout oocyte using an in vitro and in vivo study. First, synthetic single strand and natural double strand DNA from Eukaryotic and prokaryotic sources as well as naked DNA were in vitro incubated with the oocyte cytoplasm. Results showed that the DNase activity of rainbow trout oocyte is strong enough to degrade any type of DNA at the onset of the incubation. Then, we evaluated if similar to the mammalian species, dead spermatozoa from rainbow trout can protect exogenous DNA from oocyte DNases. A series of dead spermatozoa was incubated with pDB2, carrying EGFP transgene, for 30 min followed by the ooplasm treatment for an additional 30 min. Not only did oocyte DNases completely degrade the exogenous DNA, but also it degraded the compact genome of spermatozoa. In conclusion, in vitro results clearly showed that strong DNase activity of ooplasm could degrade any types of foreign DNAs including oligonucleotides and intensively compact sperm genome. The strong DNase activity of rainbow trout ooplasm could be a stumbling block for genetic modification using plasmids in salmonids.

Simultaneous effects of IGF1 and Fadrozole on parthenogenesis and pluripotency markers in chicken embryo.

So far, a synergistic effect was detected between insulin-like growth factor-I (IGF1) and anti-aromatase for sex reversal and pre-/post-natal growth of fertilized chicken embryo. Here, we hypothesized whether the growth and sexual female-to-male reversal effects of IGF1 and an anti-aromatase, Fadrozole, could improve the development of unfertilized, parthenogenetic chicken embryos. Simultaneous administration of IGF1 and Fadrozole increased the percentage of grade A embryos from 1.7% (no injection group) to 70.6%. The expression profile of parthenotes and newly laid fertilized embryos showed that IGF1 and Fadrozole increased SOX2 and NANOG expression, while decreased the TBX3 expression in the parthenogenetic embryos. However, a considerably higher expression of PRDM16, IGF2, NODAL and HDAC2 was observed in the fertilized group compared to the parthenogenetic embryos. In conclusion, chicken sexual determination is initiated at the earliest stage of embryonic development before gonadal differentiation. Combined administration of IGF1 and Fadrozole increased the developmental rate of parthenogenetic embryos. Also, simultaneous supplementation of IGF1 and Fadrozole induced the expression of pluripotency genes with no effect on the expression of growth and differentiation factors.

Expression of genes involved in BMP and estrogen signaling and AMPK production can be important factors affecting total number of antral follicles in ewes.

Follicular growth and ovulation of healthy oocytes is a complicated process which is regulated by several endocrine and paracrine factors as well as cross-talk between the oocyte and its surrounding somatic cells. This study compared the expression profile of some candidate genes involved in BMP signaling as well as estrogen and AMPK production in cumulus-oocyte complex (COC) of small and large antral follicles and their associated somatic cell layers in ovaries from ewes with high- and low-antral follicle count (AFC). Expression of GDF9 was increased by increasing the size of antral follicles, while BMP15 expression was decreased by follicular size. It should be noteworthy that transcription of both GDF9 and BMP15 was also detected in the adjacent cellular layers under the follicles. There was a very strong positive correlation between BMP15 and BMPR2 in ovary tissues. Expression of GDF9 was highly correlated with BMP15, BMPR1B, and BMPR2 in large antral follicles. Expression of BMP7 in small antral follicles and BMPR2 in ovary tissues was significantly increased in the high-AFC group. Expression of ESR1 and ESR2 involved in estrogen production as well as PRKAA1 which involved in AMPK production were significantly greater in large antral follicles of high-AFC. There was a very high correlation between Cyp19 and ESR1 in large antral follicles and ovary tissues. Expression of Cyp19 and PRKAA1 were positively correlated with GDF9, BMP15, and BMP7 in large follicles. In conclusion, this study suggests that apart from the BMP signaling, genes involved in AMPK and estrogen production can be pivotal players in ewe's follicular development process. In addition, a strong cross-talk can exist among AMPK, BMP signaling, and estrogen synthesis systems in ewe ovary.

Developing a puncture-free in ovo chicken transfection strategy based on bypassing albumen nucleases.

Chicken is a dual-purpose animal important from both agricultural and medical aspects. Even though significant improvements have been made in chicken transgenesis technologies, chicken genome manipulation has not been widely used in developmental biology. This study was aimed to evaluate chicken egg white nuclease properties and thereof plausibility of devising an in vivo transfection technology without causing physical damage to the embryo. First, the nuclease activity of egg albumen was assessed. The egg white nucleases were strongly active in degrading DNA and RNA. The egg white DNase activity was comparable to commercially available DNase-I. Nuclease activities were also assessed after heating, proteinase K, or EDTA treatment. Unlike proteinase K, both heating and EDTA were noticeably effective for the nuclease inactivation. Simultaneous application of lipoplex form of DNA (1 µg pDB2: 3 µl Lipofectamine2000) and EDTA showed a synergistic effect in protection against egg white nucleases. Finally, we injected the lipoplexes with or without EDTA close to the embryo at day0, but outside the embryonic epiblast. Implementation of a scrutinized PCR assay indicated that transfection took place only when EDTA was complemented to the lipoplexes. The transfection rate of day4 embryos and the hatched chicks were 54.5 and 30.0%, respectively. EGFP expression was detected in two out of three transgenic chicks. In conclusion, this study provided a detail analysis of chicken egg albumen nuclease properties and suggested the feasibility of developing a puncture-free handmade technology for transfection of the chicken embryo.