Comparison of ICSI, IVF, and in vivo derived embryos to produce CRISPR-Cas9 gene-edited pigs for xenotransplantation.

Genome editing in pigs for xenotransplantation has seen significant advances in recent years. This study compared three methodologies to generate gene-edited embryos, including co-injection of sperm together with the CRISPR-Cas9 system into oocytes, named ICSI-MGE (mediated gene editing); microinjection of CRISPR-Cas9 components into oocytes followed by in vitro fertilization (IVF), and microinjection of in vivo fertilized zygotes with the CRISPR-Cas9 system. Our goal was to knock-out (KO) porcine genes involved in the biosynthesis of xenoantigens responsible for the hyperacute rejection of interspecific xenografts, namely GGTA1, CMAH, and ß4GalNT2. Additionally, we attempted to KO the growth hormone receptor (GHR) gene with the aim of limiting the growth of porcine organs to a size that is physiologically suitable for human transplantation. Embryo development, pregnancy, and gene editing rates were evaluated. We found an efficient mutation of the GGTA1 gene following ICSI-MGE, comparable to the results obtained through the microinjection of oocytes followed by IVF. ICSI-MGE also showed higher rates of biallelic mutations compared to the other techniques. Five healthy piglets were born from in vivo-derived embryos, all of them exhibiting biallelic mutations in the GGTA1 gene, with three displaying mutations in the GHR gene. No mutations were observed in the CMAH and ß4GalNT2 genes. In conclusion, in vitro methodologies showed high rates of gene-edited embryos. Specifically, ICSI-MGE proved to be an efficient technique for obtaining homozygous biallelic mutated embryos. Lastly, only live births were obtained from in vivo-derived embryos showing efficient multiple gene editing for GGTA1 and GHR.

Aggregation of Leopardus geoffroyi hybrid embryos with domestic cat tetraploid blastomeres.

Heterospecific embryo transfer of an endangered species has been carried out using recipients from related domestic females. Aggregation of an embryo from an endangered species with a tetraploid embryo from the species to be transferred could improve the development of pregnancy to term. The main objective of the present study was to analyze embryo aggregation in domestic cat model using hybrid embryos. For this purpose, we compared in vitro development of synchronic (Sync) or asynchronic (Async) and asynchronic with a tetraploid (Async4n) aggregation of domestic cat IVF embryos. Furthermore, aggregated blastocyst quality was analyzed by evaluation of the total cell number, cell allocation by mitotrackers staining of embryonic cells, expression of Oct4, Nanog, Sox2, Cdx2 genes, number of OCT4+ nuclei, and presence of DNA fragmentation. Additionally, the developmental rates of Async4n aggregation of domestic cat with Leopardus geoffroyi hybrid (hLg) embryos were evaluated. Async aggregation increased blastocyst cell number and the number of OCT4+ nuclei as compared to non-aggregated diploid (2n) and tetraploid (4n) embryos. Moreover, blastocysts produced by Async4n aggregation showed reduced rates of fragmented DNA. No differences were found in the expression of the pluripotent genes, with exception of the Cdx2 expression, which was higher in 4n and aggregated embryos as compared to the control group. Interestingly, hybrids embryos derived by Async4n aggregation with domestic cat embryos had similar rates of blastocysts development as the control. Altogether, the findings support the use of two-cell-fused embryos to generate tetraploid blastomeres and demonstrate that Async4n aggregation generates good quality embryos.

CRISPR-on for activation of endogenous SMARCA4 and TFAP2C expression in bovine embryos.

CRISPR-mediated transcriptional activation, also known as CRISPR-on, has proven efficient for activation of individual or multiple endogenous gene expression in cultured cells from several species. However, the potential of CRISPR-on technology in preimplantation mammalian embryos remains to be explored. Here, we report for the first time the successful modulation of endogenous gene expression in bovine embryos by using the CRISPR-on system. As a proof of principle, we targeted the promoter region of either SMARCA4 or TFAP2C genes, transcription factors implicated in trophoblast lineage commitment during embryo development. We demonstrate that CRISPR-on provides temporal control of endogenous gene expression in bovine embryos, by simple cytoplasmic injection of CRISPR RNA components into one cell embryos. dCas9VP160 activator was efficiently delivered and accurately translated into protein, being detected in the nucleus of all microinjected blastomeres. Our approach resulted in the activation of SMARCA expression shortly after microinjection, with a consequent effect on downstream differentiation promoting factors, such as TFAP2C and CDX2. Although targeting of TFAP2C gene did not result in a significant increase in TFAP2C expression, there was a profound induction in CDX2 expression on day 2 of development. Finally, we demonstrate that CRISPR-on system is suitable for gene expression modulation during the preimplantation period, since no detrimental effect was observed on microinjected embryo development. This study constitutes a first step toward the application of the CRISPR-on system for the study of early embryo cell fate decisions in cattle and other mammalian embryos, as well as to design novel strategies that may lead to an improved trophectoderm development.

Practical Approaches for Knock-Out Gene Editing in Pigs.

Pigs are an important resource for meat production and serve as a model for human diseases. Due to their physiological and anatomical similarities to humans, these animals can recapitulate symptoms of human diseases, becoming an effective model for biomedical research. Although, in the past pig have not been widely used partially because of the difficulty in genetic modification; nowadays, with the new revolutionary technology of programmable nucleases, and fundamentally of the CRISPR-Cas9 systems, it is possible for the first time to precisely modify the porcine genome as never before. To this purpose, it is necessary to introduce the system into early stage zygotes or to edit cells followed by somatic cell nuclear transfer. In this review, several strategies for pig knock-out gene editing, using the CRISPR-Cas9 system, will be summarized, as well as genotyping methods and different delivery techniques to introduce these tools into the embryos. Finally, the best approaches to produce homogeneous, biallelic edited animals will be discussed.

A eukaryotic nicotinate-inducible gene cluster: convergent evolution in fungi and bacteria.

Nicotinate degradation has hitherto been elucidated only in bacteria. In the ascomycete Aspergillus nidulans, six loci, hxnS/AN9178 encoding the molybdenum cofactor-containing nicotinate hydroxylase, AN11197 encoding a Cys2/His2 zinc finger regulator HxnR, together with AN11196/hxnZ, AN11188/hxnY, AN11189/hxnP and AN9177/hxnT, are clustered and stringently co-induced by a nicotinate derivative and subject to nitrogen metabolite repression mediated by the GATA factor AreA. These genes are strictly co-regulated by HxnR. Within the hxnR gene, constitutive mutations map in two discrete regions. Aspergillus nidulans is capable of using nicotinate and its oxidation products 6-hydroxynicotinic acid and 2,5-dihydroxypyridine as sole nitrogen sources in an HxnR-dependent way. HxnS is highly similar to HxA, the canonical xanthine dehydrogenase (XDH), and has originated by gene duplication, preceding the origin of the Pezizomycotina. This cluster is conserved with some variations throughout the Aspergillaceae. Our results imply that a fungal pathway has arisen independently from bacterial ones. Significantly, the neo-functionalization of XDH into nicotinate hydroxylase has occurred independently from analogous events in bacteria. This work describes for the first time a gene cluster involved in nicotinate catabolism in a eukaryote and has relevance for the formation and evolution of co-regulated primary metabolic gene clusters and the microbial degradation of N-heterocyclic compounds.

Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.

Bovine parthenogenotes produced by inhibition of first or second polar bodies emission.

Parthenogenetic embryos are an ethically acceptable alternative for the derivation of human embryonic stem cells. In this work, we propose a new strategy to produce bovine parthenogenetic embryos inhibiting the emission of the first polar body during in vitro maturation, and allowing the extrusion of the second polar body during oocyte activation. Cytochalasin B, an inhibitor of actin microfilaments, was employed during in vitro maturation to inhibit first polar body emission or during parthenogenetic activation to block second polar body emission. Only one polar body was inhibited in each strategy in order to keep the diploid chromosome set. In experiment 1, the effect of cytochalasin B on in vitro maturation of bovine oocytes was evaluated. Most oocytes (77%) were arrested at a meiotic stage characterized by the presence of a large internal metaphase plate and absence of polar body. In experiment 2, development of embryos exposed to cytochalasin B during in vitro maturation (CytoB-IVM) or during activation (CytoB-ACT) was compared. Developmental rates did not differ between diploidization strategies, even when three agents were employed to induce activation. Both groups, CytoB-IVM and CytoB-ACT, tended to maintain diploidy. CytoB-IVM parthenogenesis could help to obtain embryos with a higher degree of homology to the oocyte donor.

Bovine parthenogenotes produced by inhibition of first or second polar bodies emission

Parthenogenetic embryos are an ethically acceptable alternative for the derivation of human embryonic stem cells. In this work, we propose a new strategy to produce bovine parthenogenetic embryos inhibiting the emission of the first polar body during in vitro maturation, and allowing the extrusion of the second polar body during oocyte activation. Cytochalasin B, an inhibitor of actin microfilaments, was employed during in vitro maturation to inhibit first polar body emission or during parthenogenetic activation to block second polar body emission. Only one polar body was inhibited in each strategy in order to keep the diploid chromosome set. In experiment 1, the effect of cytochalasin B on in vitro maturation of bovine oocytes was evaluated. Most oocytes (77%) were arrested at a meiotic stage characterized by the presence of a large internal metaphase plate and absence of polar body. In experiment 2, development of embryos exposed to cytochalasin B during in vitro maturation (CytoB-IVM) or during activation (CytoB-ACT) was compared. Developmental rates did not differ between diploidization strategies, even when three agents were employed to induce activation. Both groups, CytoB-IVM and CytoB-ACT, tended to maintain diploidy. CytoB-IVM parthenogenesis could help to obtain embryos with a higher degree of homology to the oocyte donor.

Novel methods to induce exogenous gene expression in SCNT, parthenogenic and IVF preimplantation bovine embryos.

The import of exogenous DNA (eDNA) from the cytoplasm to the nucleus represents a key intracellular obstacle for efficient gene delivery in mammalian cells. In this study, cumulus cells or oolemma vesicles previously incubated with eDNA, and naked eDNA were injected into the cytoplasm of MII oocytes to evaluate their efficiency for eDNA expressing bovine embryo production. Our study evaluated the potential of short time co-incubation (5 min) of eDNA with; (1) cumulus cells, to be used as donor cells for SCNT and (2) oolemma vesicles (vesicles) to produce parthenogenic transgene expressing embryos. In addition, we included a group consisting of the injection of eDNA alone (plasmid) followed by parthenogenic activation. Two different pCX-EGFP plasmid concentrations (50 and 500 ng/µl) were employed. The results showed that embryos produced by SCNT and by vesicle injection assisted by chemical activation were able to express the eDNA in higher rates than embryos injected with plasmid alone. The lower plasmid concentration allowed the highest development rates in all groups. Using confocal microscopy, we analyzed the interaction of FITC- labeled eDNA with cumulus cells and vesicles as well as oocytes injected with labeled plasmid alone. Our images demonstrated that eDNA interacted with cumulus cells and vesicles, resulting an increase in its expression efficiency. In contrast, oocytes injected with DNA alone did not show signs of transgene accumulation, and their eDNA expression rates were lower. In a further experiment, we evaluated if transgene-expressing embryos could be produced by means of vesicle injection followed by IVF. The lower plasmid concentration (50 ng/µl) injected after IVF, produced the best results. Preliminary FISH analysis indicated detectable integration events in 1/5 of SCNT blastocysts treated. Our studies demonstrate for the first time that short term transgene co-incubation with somatic cells can produce transgene-expressing mammalian SCNT embryos and also that parthenogenic, eDNA- expressing embryos can be obtained by injection of vesicles or eDNA alone. Moreover, eDNA-expressing embryos can be also obtained by cytoplasmic injection vesicles in IVF zygotes, simplifying the traditional IVF pronuclear injection technique.

Effects of bone morphogenic protein 4 (BMP4) and its inhibitor, Noggin, on in vitro maturation and culture of bovine preimplantation embryos.

BACKGROUND: BMP4 is a member of the transforming growth factor beta (TGFbeta) superfamily and Noggin is a potent BMP inhibitor that exerts its function by binding to BMPs preventing interactions with its receptors. The aim of this work was to investigate the role of BMP4 and Noggin, on oocytes in vitro maturation (m experiments) and embryos in vitro development (c experiments) of bovine. METHODS: For m experiments, COCs were collected from slaughterhouse ovaries and in vitro matured in TCM with 100 ng/ml of either BMP4 or Noggin. After 24 h, the nuclear stage of the oocytes was determined by staining with Hoechst 33342. In addition, RT-qPCR was performed on MII oocytes to study the relative concentration of ZAR1, GDF9, BAX, MATER and HSP70 transcripts. Treated oocytes were submitted to parthenogenic activation (PA) or in vitro fertilization (IVF) and cultured in CR2. For c experiments, non-treated matured oocytes were submitted to PA or IVF to generate embryos that were exposed to 100 ng/ml of BMP4 or Noggin in CR2 until day nine of culture. Cleavage, blastocyst and hatching rates, expression pattern of the transcription factor Oct-4 in blastocysts and embryo cell number at day two and nine post-activation or fertilization were evaluated. RESULTS: We found that Noggin, as BMP4, did not affect oocyte nuclear maturation. Noggin supplementation up-regulated the expression of HSP70 and MATER genes in matured oocytes. Moreover, BMP4 during maturation increased the proportion of Oct-4 positive cells in parthenogenic embryos. On the other hand, when Noggin was added to embryo culture medium, developmental rates of parthenogenic and in vitro fertilized embryos were reduced. However, BMP4 addition decreases the development only for in vitro fertilized embryos. BMP4 and Noggin during culture reduced the proportion of Oct-4-expressing cells. CONCLUSIONS: Our results show that BMP4 is implicated in bovine oocytes maturation and embryo development. Moreover, our findings demonstrate, for the first time, that a correct balance of BMP signaling is needed for proper pre-implantation development of bovine embryos.

Efficiency of sperm-mediated gene transfer in the ovine by laparoscopic insemination, in vitro fertilization and ICSI.

Transgenesis constitutes an important tool for pharmacological protein production and livestock improvement. We evaluated the potential of laparoscopic insemination (LI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to produce egfp-expressing ovine embryos, using spermatozoa previously exposed to pCX-EGFP plasmid in two different sperm/DNA incubation treatments: "Long Incubation" (2 h at 17 C) and "Short Incubation" (5 min at 5 C). For LI, Merino sheep were superovulated and inseminated with treated fresh semen from Merino rams. The embryos were recovered by flushing the uterine horns. For IVF and ICSI, slaughterhouse oocytes were fertilized with DNA-treated frozen/thawed sperm. All recovered embryos were exposed to blue light (488 nm) to determine green fluorescent morulae and blastocysts rates. High cleavage and morulae/blastocysts rates accompanied the LI and IVF procedures, but no egfp-expressing embryos resulted. In contrast, regardless of the sperm/plasmid incubation treatment, egfp-expressing morulae and blastocysts were always obtained by ICSI, and the highest transgenesis rate (91.6%) was achieved with Short Incubation. In addition, following the incubation of labeled plasmid DNA, after Long or Short exposure treatments, with fresh or frozen/thawed spermatozoa, only non-motile fresh spermatozoa could maintain an attached plasmid after washing procedures. No amplification product could be detected following PCR treatment of LI embryos whose zonae pellucidae (ZP) had been removed. In order to establish conditions for transgenic ICSI in the ovine, we compared three different activation treatments, and over 60% of the obtained blastocysts expressed the transgene. For ICSI embryos, FISH analysis found possible signals compatible with integration events. In conclusion, our results show that in the ovine, under the conditions studied, ICSI is the only method capable of producing exogenous gene-expressing embryos using spermatozoa as vectors.

Activation with ionomycin followed by dehydroleucodine and cytochalasin B for the production of parthenogenetic and cloned bovine embryos.

In this work, Dehydroleucodine (DhL) was evaluated as a chemical activator of bovine oocytes and somatic cell nuclear transfer (SCNT) reconstituted embryos. Oocytes were activated with 5 microM Ionomycin (Io) and exposed for 3 h to 1 or 5 microM DhL alone (Io-Dhl1 or Io-DhL5) or combined with Cytochalasin B (Io-DhL1/CB; Io-DhL5/CB). Control groups were Io (Io), Io followed by 1.9 mM 6-Dimethylaminopurine (Io-6DMAP), and embryos produced by in vitro fertilization (IVF). Pronuclear formation and development to blastocysts of activated oocytes were evaluated. Embryos obtained by the DhL concentration that induced the highest blastocyst rates (1 microM) were karyotyped. An additional treatment based in Io-DhL1 plus lengthened (6-h) exposure to CB (Io-DhL1/long CB) was included to improve the proportion of diploid blastomeres. Finally, DhL combined with CB was employed to assist cloning by intracytoplasmic injection of whole cumulus cells. Results showed that DhL induces a pronuclear formation dynamic that was more similar to IVF-produced embryos than DMAP. Development to blastocyst stage was higher after activation with 1 microM DhL than with 5 microM DhL, either for groups combined or not with CB (19.15; 21.74 vs. 6.82; 0%, respectively) (p < 0.05). Io-DhL1 and Io-DhL1/CB treatments induced blastocyst-cleaved embryo ratios not statistically different from those of Io-DMAP (35.85%) and IVF (33.33%) groups (p > 0.05). Io-DhL1/long CB induced higher diploid blastomere rates than Io-Dhl1, Io-DhL1/CB and Io-DMAP (63.8 vs. 36.8; 40 and 31.6%, respectively) (p < 0.05). Moreover, all DhL treatments resulted in polyploidy rates that were lower than Io-DMAP (5.2, 12.0, 10.6, and 31.6%, respectively) (p < 0.05). Io-DhL1/CB and Io-DhL1/long CB induced cloned embryo blastocyst rates that were not significantly different from Io-DMAP (6.1, 9.4, and 18.3%, respectively) (p < 0.05). Our results indicate that Io-DhL1/long CB protocol could be useful for SCNT programs.

High rates of bovine blastocyst development after ICSI-mediated gene transfer assisted by chemical activation.

In order to establish conditions for intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) in cattle, various aspects of fertilization and embryonic development were assessed after five activation treatments. Spermatozoa were co-incubated with pCX-EGFP (pCX-enhanced green fluorescent protein gene) plasmid and injected into metaphase II oocytes, which were then treated with ionomycin (Io), before further activation with the following agents: 6-dimethylaminopurine (Io-DMAP), additional Io plus DMAP (2Io-DMAP), Io alone (2Io), ethanol (Io-EtOH), or strontium chloride (Io-SrCl2). Fertilization rates at 16 h after ICSI, presence of a condensed spermatozoon head on Day 4 (Day 0 = ICSI), blastocyst and EGFP expression rates on Day 7, and Oct-4 pattern of Day 8 blastocysts were evaluated. Fertilization rates did not differ significantly among treatments. All (100%) of EGFP-positive embryos resulted from ICSI fertilization, whereas at least 60% of EGFP-negative embryos (>4 cells) had a condensed sperm head. Blastocyst rates after 2Io-DMAP were not significantly different from Io-DMAP or Io-EtOH, but they were higher than 2Io or Io-SrCl2 treatments (25.9, 18.7, 14.7, 9.4, and 10.9% respectively; P < 0.05). Transgene expression rates were higher for Io-DMAP, 2Io-DMAP and Io-SrCl2 than for 2Io and Io-EtOH (52.3, 53.0, 42.8, 28.2, and 29.4% respectively; P < 0.05). Over 80% of the blastocysts expressed egfp protein. In conclusion, ICSI-MGT was a powerful technique to produce bovine embryos that expressed the EGFP transgene. Moreover, the actual efficiency of ICSI-MGT could be readily evaluated by this method, which uses a marker expressed early in embryo development.

Nucleosome positioning and histone H3 acetylation are independent processes in the Aspergillus nidulans prnD-prnB bidirectional promoter.

In Aspergillus nidulans, proline can be used as a carbon and nitrogen source, and its metabolism requires the integration of three signals, including proline induction and nitrogen and carbon metabolite derepression. We have previously shown that the bidirectional promoter in the prnD-prnB intergenic region undergoes drastic chromatin rearrangements such that proline induction leads to the loss of positioned nucleosomes, whereas simultaneous carbon and nitrogen metabolite repression results in the partial repositioning of these nucleosomes. In the proline cluster, the inhibition of deacetylases by trichostatin A leads to partial derepression and is associated with a lack of nucleosome positioning. Here, we investigate the effect of histone acetylation in the proline cluster using strains deleted of essential components of putative A. nidulans histone acetyltransferase complexes, namely, gcnE and adaB, the orthologues of the Saccharomyces cerevisiae GCN5 and ADA2 genes, respectively. Surprisingly, GcnE and AdaB are not required for transcriptional activation and chromatin remodeling but are required for the repression of prnB and prnD and for the repositioning of nucleosomes in the divergent promoter region. Chromatin immunoprecipitation directed against histone H3 lysines K9 and K14 revealed that GcnE and AdaB participate in increasing the acetylation level of at least one nucleosome in the prnD-prnB intergenic region during activation, but these activities do not determine nucleosome positioning. Our results are consistent with a function of GcnE and AdaB in gene repression of the proline cluster, probably an indirect effect related to the function of CreA, the DNA-binding protein mediating carbon catabolite repression in A. nidulans.

The tightly regulated promoter of the xanA gene of Aspergillus nidulans is included in a helitron.

In Aspergillus nidulans the xanA gene codes for a xanthine alpha-ketoglutarate-dependent dioxygenase, an enzyme only present in the fungal kingdom. The 5' region of this gene, including its putative promoter and the first 54 codons of the open reading frame, together with the first intron is duplicated in the genome. This duplication corresponds to a helitron, a eukaryotic element proposed to transpose replicatively by the rolling circle mechanism. We show that the regulation of xanA conforms to that of other genes of the purine degradation pathway, necessitating the specific UaY transcription factor and the AreA GATA factor. The promoter of the duplicated region is active ectopically and the difficulty in detecting an mRNA from the duplicated region is at least partially due to nonsense-mediated decay. Comparative genomic data are only consistent with the hypothesis that the 5' region of xanA pre-existed the helitron insertion, and that a 'secondary helitron' was generated from an insertion 5' to it and a pre-existing 3' consensus sequence within the open reading frame. It is possible to propose a role of helitrons in promoter shuffling and thus in recruiting new genes into specific regulatory circuits.

Convergent evolution of hydroxylation mechanisms in the fungal kingdom: molybdenum cofactor-independent hydroxylation of xanthine via alpha-ketoglutarate-dependent dioxygenases.

The xanthine oxidases and dehydrogenases are among the most conserved enzymes in all living kingdoms. They contain the molybdopterin cofactor Moco. We show here that in the fungi, in addition to xanthine dehydrogenase, a completely different enzyme is able to catalyse the oxidation of xanthine to uric acid. In Aspergillus nidulans this enzyme is coded by the xanA gene. We have cloned the xanA gene and determined its sequence. A deletion of the gene has the same phenotype as the previously known xanA1 miss-sense mutation. Homologues of xanA exist only in the fungal kingdom. We have inactivated the cognate gene of Schizosaccharomyces pombe and this results in strongly impaired xanthine utilization as a nitrogen source. We have shown that the Neurospora crassa homologue is functionally equivalent to xanA. The enzyme coded by xanA is an alpha-ketoglutarate- and Fe(II)-dependent dioxygenase which shares a number of properties with other enzymes of this group. This work shows that only in the fungal kingdom, an alternative mechanism of xanthine oxidation, not involving Moco, has evolved using the dioxygenase scaffold.

A gene of the opsin family in the carotenoid gene cluster of Fusarium fujikuroi.

Opsins are membrane photoreceptors closely related to the heat-shock proteins of the HSP30 family. Their functions include light-driven ion pumping in archaea and light detection in algae and animals, using the apocarotenoid retinal as a light-absorbing prosthetic group. We describe a gene of Fusarium fujikuroi, carO, coding for a polypeptide resembling opsins and HSP30-like proteins and contiguous to the genes of the carotenoid pathway, carRA and carB. Transcription of carO is induced by light and is deregulated in carotenoid-overproducing mutants. The same regulation pattern is exhibited by carRA and carB; and common conserved DNA elements are found in the three promoters. Heat shock resulted in a modest induction of carO transcription, similar to the one exhibited by carB, confirming a common regulation. Targeted mutagenesis of carO produced no apparent phenotypic modification, including no change in the photoinduction of carotenoid biosynthesis.