In vivo CRISPR/LbCas12a-mediated knock-in and knock-out in Atlantic salmon (Salmo salar L.).

Genome editing using the CRISPR/Cas system offers the potential to enhance current breeding programs and introduce desirable genetic traits, including disease resistance, in salmon aquaculture. Several nucleases are available using this system, displaying differences regarding structure, cleavage, and PAM requirement. Cas9 is well established in Atlantic salmon, but Cas12a has yet to be tested in vivo in this species. In the present work, we microinjected salmon embryos with LbCas12a ribonucleoprotein complexes targeting the pigmentation gene solute carrier family 45 member 2 (slc45a2). Using CRISPR/LbCas12a, we were able to knock-out slc45a2 and knock-in a FLAG sequence element by providing single-stranded DNA templates. High-throughput sequencing revealed perfect HDR rates up to 34.3% and 54.9% in individual larvae using either target or non-target strand template design, respectively. In this work, we demonstrate the in vivo application of CRISPR/LbCas12a in Atlantic salmon, expanding the toolbox for editing the genome of this important aquaculture species.

Single nucleotide replacement in the Atlantic salmon genome using CRISPR/Cas9 and asymmetrical oligonucleotide donors.

BACKGROUND: New breeding technologies (NBT) using CRISPR/Cas9-induced homology directed repair (HDR) has the potential to expedite genetic improvement in aquaculture. The long generation time in Atlantic salmon makes breeding an unattractive solution to obtain homozygous mutants and improving the rates of perfect HDR in founder (F0) fish is thus required. Genome editing can represent small DNA changes down to single nucleotide replacements (SNR). This enables edits such as premature stop codons or single amino acid changes and may be used to obtain fish with traits favorable to aquaculture, e.g. disease resistance. A method for SNR has not yet been demonstrated in salmon. RESULTS: Using CRISPR/Cas9 and asymmetrical ODNs, we were able to perform precise SNR and introduce a premature stop codon in dnd in F0 salmon. Deep sequencing demonstrated up to 59.2% efficiency in single embryos. In addition, using the same asymmetrical ODN design, we inserted a FLAG element into slc45a2 and dnd, showing high individual perfect HDR efficiencies (up to 36.7 and 32.7%, respectively). CONCLUSIONS: In this work, we demonstrate that precise SNR and knock-in (KI) can be performed in F0 salmon embryos using asymmetrical oligonucleotide (ODN) donors. We suggest that HDR-induced SNR can be applied as a powerful NBT, allowing efficient introgression of favorable alleles and bypassing challenges associated with traditional selective breeding.

Correction: The novel microRNAs hsa-miR-nov7 and hsa-miR-nov3 are over-expressed in locally advanced breast cancer.

[This corrects the article DOI: 10.1371/journal.pone.0225357.].

Rescue of germ cells in dnd crispant embryos opens the possibility to produce inherited sterility in Atlantic salmon.

Genetic introgression of escaped farmed Atlantic salmon (Salmo salar) into wild populations is a major environmental concern for the salmon aquaculture industry. Using sterile fish in commercial aquaculture operations is, therefore, a sustainable strategy for bio-containment. So far, the only commercially used methodology for producing sterile fish is triploidization. However, triploid fish are less robust. A novel approach in which to achieve sterility is to produce germ cell-free salmon, which can be accomplished by knocking out the dead-end (dnd) gene using CRISPR-Cas9. The lack of germ cells in the resulting dnd crispants, thus, prevents reproduction and inhibits subsequent large-scale production of sterile fish. Here, we report a rescue approach for producing germ cells in Atlantic salmon dnd crispants. To achieve this, we co-injected the wild-type (wt) variant of salmon dnd mRNA together with CRISPR-Cas9 constructs targeting dnd into 1-cell stage embryos. We found that rescued one-year-old fish contained germ cells, type A spermatogonia in males and previtellogenic primary oocytes in females. The method presented here opens a possibility for large-scale production of germ-cell free Atlantic salmon offspring through the genetically sterile broodstock which can pass the sterility trait on the next generation.

The novel microRNAs hsa-miR-nov7 and hsa-miR-nov3 are over-expressed in locally advanced breast cancer.

miRNAs are an important class of small non-coding RNAs, which play a versatile role in gene regulation at the post-transcriptional level. Expression of miRNAs is often deregulated in human cancers. We analyzed small RNA massive parallel sequencing data from 50 locally advanced breast cancers aiming to identify novel breast cancer related miRNAs. We successfully predicted 10 novel miRNAs, out of which 2 (hsa-miR-nov3 and hsa-miR-nov7) were recurrent. Applying high sensitivity qPCR, we detected these two microRNAs in 206 and 214 out of 223 patients in the study from which the initial cohort of 50 samples were drawn. We found hsa-miR-nov3 and hsa-miR-nov7 both to be overexpressed in tumor versus normal breast tissue in a separate set of 13 patients (p = 0.009 and p = 0.016, respectively) from whom both tumor tissue and normal tissue were available. We observed hsa-miR-nov3 to be expressed at higher levels in ER-positive compared to ER-negative tumors (p = 0.037). Further stratifications revealed particularly low levels in the her2-like and basal-like cancers compared to other subtypes (p = 0.009 and 0.040, respectively). We predicted target genes for the 2 microRNAs and identified inversely correlated genes in mRNA expression array data available from 203 out of the 223 patients. Applying the KEGG and GO annotations to target genes revealed pathways essential to cell development, communication and homeostasis. Although a weak association between high expression levels of hsa-miR-nov7 and poor survival was observed, this did not reach statistical significance. hsa-miR-nov3 expression levels had no impact on patient survival.

Indel locations are determined by template polarity in highly efficient in vivo CRISPR/Cas9-mediated HDR in Atlantic salmon.

Precise gene editing such as CRISPR/Cas9-mediated homology directed repair (HDR) can increase our understanding of gene function and improve traits of importance for aquaculture. This fine-tuned technology has not been developed for farmed fish including Atlantic salmon. We performed knock-in (KI) of a FLAG element in the slc45a2 gene in salmon using sense (S), anti-sense (AS) and double-stranded (ds) oligodeoxynucleotide (ODN) templates with short (24/48/84 bp) homology arms. We show in vivo ODN integration in almost all the gene edited animals, and demonstrate perfect HDR rates up to 27% in individual F0 embryos, much higher than reported previously in any fish. HDR efficiency was dependent on template concentration, but not homology arm length. Analysis of imperfect HDR variants suggest that repair occurs by synthesis-dependent strand annealing (SDSA), as we show for the first time in any species that indel location is dependent on template polarity. Correct ODN polarity can be used to avoid 5'-indels interrupting the reading frame of an inserted sequence and be of importance for HDR template design in general.

MYC-Induced miR-203b-3p and miR-203a-3p Control Bcl-xL Expression and Paclitaxel Sensitivity in Tumor Cells.

Taxanes are chemotherapeutic agents used in the treatment of solid tumors, particularly of breast, ovarian, and lung origin. However, patients show divergent therapy responses, and the molecular determinants of taxane sensitivity have remained elusive. Especially the signaling pathways that promote death of the taxane-treated cells are poorly characterized. Here we describe a novel part of a signaling route in which c-Myc enhances paclitaxel sensitivity through upregulation of miR-203b-3p and miR-203a-3p; two clustered antiapoptosis protein Bcl-xL controlling microRNAs. In vitro, the miR-203b-3p decreases the expression of Bcl-xL by direct targeting of the gene's mRNA 3'UTR. Notably, overexpression of the miR-203b-3p changed the fate of paclitaxel-treated breast and ovarian cancer cells from mitotic slippage to cell death. In breast tumors, high expression of the miR-203b-3p and MYC was associated with better therapy response and patient survival. Interestingly, in the breast tumors, MYC expression correlated negatively with BCL2L1 expression but positively with miR-203b-3p and miR-203a-3p. Finally, silencing of MYC suppressed the transcription of both miRNAs in breast tumor cells. Pending further validation, these results may assist in patient stratification for taxane therapy.

Novel Mad2-targeting miR-493-3p controls mitotic fidelity and cancer cells' sensitivity to paclitaxel.

The molecular pathways that contribute to the proliferation and drug response of cancer cells are highly complex and currently insufficiently characterized. We have identified a previously unknown microRNA-based mechanism that provides cancer cells means to stimulate tumorigenesis via increased genomic instability and, at the same time, evade the action of clinically utilized microtubule drugs. We demonstrate miR-493-3p to be a novel negative regulator of mitotic arrest deficient-2 (MAD2), an essential component of the spindle assembly checkpoint that monitors the fidelity of chromosome segregation. The microRNA targets the 3' UTR of Mad2 mRNA thereby preventing translation of the Mad2 protein. In cancer cells, overexpression of miR-493-3p induced a premature mitotic exit that led to increased frequency of aneuploidy and cellular senescence in the progeny cells. Importantly, excess of the miR-493-3p conferred resistance of cancer cells to microtubule drugs. In human neoplasms, miR-493-3p and Mad2 expression alterations correlated with advanced ovarian cancer forms and high miR-493-3p levels were associated with reduced survival of ovarian and breast cancer patients with aggressive tumors, especially in the paclitaxel therapy arm. Our results suggest that intratumoral profiling of miR-493-3p and Mad2 levels can have diagnostic value in predicting the efficacy of taxane chemotherapy.

Effects of SNP variants in the 17ß-HSD2 and 17ß-HSD7 genes and 17ß-HSD7 copy number on gene transcript and estradiol levels in breast cancer tissue.

Breast cancers reveal elevated E2 levels compared to plasma and normal breast tissue. Previously, we reported intra-tumour E2 to be negatively correlated to transcription levels of 17ß-HSD2 but positively correlated to 17ß-HSD7. Here, we explored these mechanisms further by analysing the same breast tumours for 17ß-HSD2 and -7 SNPs, as well as 17ß-HSD7 gene copy number. Among the SNPs detected, we found the 17ß-HSD2 rs4445895_T allele to be associated with lower intra-tumour mRNA (p=0.039) and an elevated intra-tumour E2 level (p=0.006). In contrast, we found the 17ß-HSD7 rs1704754_C allele to be associated with elevated mRNA (p=0.050) but not to E2 levels in breast tumour tissue. Surprisingly, 17ß-HSD7 - gene copy number was elevated in 19 out of 46 breast tumours examined. Elevated copy number was associated with an increased mRNA expression level (p=0.013) and elevated tumour E2 (p=0.025). Interestingly, elevated 17ß-HSD7 - gene copy number was associated with increased expression not only of 17ß-HSD7, but the 17ß-HSD7_II pseudogene as well (p=0.019). Expression level of 17ß-HSD7 and its pseudogene was significantly correlated both in tumour tissue (rs=0.457, p=0.001) and in normal tissue (rs=0.453, p=0.002). While in vitro transfection experiments revealed no direct impact of 17ß-HSD7 expression on pseudogene level, the fact that 17ß-HSD7 and 17ß-HSD7_II share a 95.6% sequence identity suggests the two transcripts may be subject to common regulatory mechanisms. In conclusion, genetic variants of 17ß-HSD2 and 17ß-HSD7 may affect intra-tumour gene expression as well as breast cancer E2 levels in postmenopausal women.

Elevated levels of the steroidogenic factor 1 are associated with over-expression of CYP19 in an oestrogen-producing testicular Leydig cell tumour.

BACKGROUND AND OBJECTIVES: Testicular Leydig cell tumours (LCTs) are rare, steroid-secreting tumours. Elevated levels of aromatase (CYP19 or CYP19A1) mRNA have been previously described in LCTs; however, little is known about the mechanism(s) causing CYP19 over-expression. We report an LCT in a 29-year-old male with elevated plasma oestradiol caused by enhanced CYP19 transcription. DESIGN AND METHODS: First, we measured the intra-tumour expression of CYP19 and determined the use of CYP19 promoters by qPCR. Secondly, we explored CYP19 and promoter II (PII) for gene amplifications and activating mutations in PII by sequencing. Thirdly, we analysed intra-tumour expression of steroidogenic factor 1 (SF-1 (NR5A1)), liver receptor homologue-1 (LRH-1 (NR5A2)) and cyclooxygenase-2 (COX2 (PTGS2)). Finally, we analysed SF-1 for promoter mutations and gene amplifications. RESULTS: Similar to what has been recorded in normal Leydig cells, we first found the bulk of tumour CYP19 transcripts to be PII derived, excluding promoter shift as a cause of enhanced transcription. Secondly, we excluded CYP19 and PII gene amplifications, and activating mutations in PII, as causes of elevated CYP19 mRNA. We found SF-1 mRNA to be up-regulated in the tumour, while LRH-1 and COX2 were down-regulated. The finding of elevated SF-1 levels in the tumour was confirmed by immunohistochemistry. The elevated level of SF-1 was not due to promoter mutations or amplifications of the SF-1 gene. CONCLUSIONS: Our results strongly suggest that the elevated levels of SF-1 have induced PII-regulated CYP19 transcription in this tumour. These findings are of relevance to the understanding of CYP19 up-regulation in general, which may occur in several tissues, including breast cancer.

Effect of CYP19 rs6493497 and rs7176005 haplotype status on in vivo aromatase transcription, plasma and tissue estrogen levels in postmenopausal women.

High plasma levels of estradiol (E(2)) are correlated to increased breast cancer risk in postmenopausal women. Recently, a study reported two single nucleotide polymorphisms (SNPs) (rs6493497; C→T and rs7176005; G→A) in the aromatase (CYP19) promoter 1.1 to be associated with elevated plasma E(2) levels, most likely due to enhanced transcription. In silico predictions suggested increased transcription factor binding for the rs7176005_A allele. We genotyped 46 breast cancer patients for rs6493497 and rs7176005 status and assessed the potential association between CYP19 SNP status and (i) CYP19 mRNA levels in tumour and normal breast tissue, and (ii) estrogen levels in plasma, tumour and normal breast tissue. In addition, we measured CYP19 SNP status and correlated it to plasma estrogen levels in a confirmatory dataset of 108 healthy postmenopausal women. We found no correlation between either of the two SNPs and CYP19 mRNA level. In the breast cancer patients, the rs6493497_T/rs7176005_A variant haplotype was associated with low plasma estrone (E(1)) (p=0.038) and low E(2) (p=0.050) levels; however, no correlation was recorded between SNP status and plasma estrogen levels in the cohort of 108 healthy postmenopausal women. Our findings indicate rs6493497 and rs7176005 status not to enhance CYP19 transcription or increase estrogen levels in postmenopausal women.

Intratumoral estrogen disposition in breast cancer.

PURPOSE: The concentration of estradiol (E(2)) in breast tumors is significantly higher than that in plasma, particularly in postmenopausal women. The contribution of local E(2) synthesis versus uptake of E(2) from the circulation is controversial. Our aim was to identify possible determinants of intratumoral E(2) levels in breast cancer patients. EXPERIMENTAL DESIGN: The expression of genes involved in estrogen synthesis, metabolism, and signaling was measured in 34 matched samples of breast tumor and normal breast tissue, and their correlation with estrogen concentrations assessed. RESULTS: ESR1 (9.1-fold; P < 0.001) and HSD17B7 (3.5-fold; P < 0.001) were upregulated in ER(+) tumors compared with normal tissues, whereas STS (0.34-fold; P < 0.001) and HSD17B5 (0.23-fold; P < 0.001) were downregulated. Intratumoral E(2) levels showed a strong positive correlation with ESR1 expression in all patients (Spearman r = 0.55, P < 0.001) and among the subgroups of postmenopausal (r = 0.76, P < 0.001; n = 23) and postmenopausal ER(+) patients (r = 0.59, P = 0.013; n = 17). HSD17B7 expression showed a significant positive correlation (r = 0.59, P < 0.001) whereas HSD17B2 (r = -0.46, P = 0.0057) and HSD17B12 (r = -0.45, P = 0.0076) showed significant negative correlations with intratumoral E(2) in all patients. Intratumoral E(2) revealed no correlation to CYP19, STS, and HSD17B1 expression. Multivariate models comprising ESR1 and plasma E(2) predicted between 50% and 70% of intratumoral E(2) variability. CONCLUSION: Uptake due to binding to the ER, rather than intratumoral estrogen synthesis by aromatase or sulfatase, is the single most important correlate and a probable determinant of intratumoral E(2). An increased expression of HSD17B7 may explain the increased ratio of E(2) to estrone (E(1)) in breast tumors compared with normal tissue.

Chk2 splice variants express a dominant-negative effect on the wild-type Chk2 kinase activity.

While the majority of RNA transcripts from protein-encoding genes in the human genome are subject to physiological splicing, pathological splicing is increasingly reported in cancer tissue. Previously, we identified >90 different splice variants of Chk2, a gene encoding a serine/threonine kinase propagating the DNA damage signal by phosphorylating and activating several downstream substrates like p53, Cdc25A, and Cdc25C involved in cell cycle arrest and apoptosis. While alternative splice forms of other genes have been reported to exert a dominant-negative effect on the wild-type molecules, the function of Chk2 splice protein variants is still unclear. Here we evaluated the function of four Chk2 splice proteins for which mRNA splice variants were identified in human breast carcinomas. These splice variants were stably expressed as nuclear proteins. Two splice forms (Chk2Delta4 and Chk2del(2-3)) expressed kinase activity while variants Chk2Delta11 and Chk2isoI were essentially kinase inactive. Independent of intrinsic kinase activity, each splice variant impaired wild-type Chk2 activity through heterodimerization. Based on our findings, we suggest alternative splicing as a possible novel mechanism for repression of the Chk2 wild-type function.

Analysing the outer membrane subproteome of Methylococcus capsulatus (Bath) using proteomics and novel biocomputing tools.

High-resolution two-dimensional gel electrophoresis and mass spectrometry has been used to identify the outer membrane (OM) subproteome of the Gram-negative bacterium Methylococcus capsulatus (Bath). Twenty-eight unique polypeptide sequences were identified from protein samples enriched in OMs. Only six of these polypeptides had previously been identified. The predictions from novel bioinformatic methods predicting beta-barrel outer membrane proteins (OMPs) and OM lipoproteins were compared to proteins identified experimentally. BOMP ( http://www.bioinfo.no/tools/bomp ) predicted 43 beta-barrel OMPs (1.45%) from the 2,959 annotated open reading frames. This was a lower percentage than predicted from other Gram-negative proteomes (1.8-3%). More than half of the predicted BOMPs in M. capsulatus were annotated as (conserved) hypothetical proteins with significant similarity to very few sequences in Swiss-Prot or TrEMBL. The experimental data and the computer predictions indicated that the protein composition of the M. capsulatus OM subproteome was different from that of other Gram-negative bacteria studied in a similar manner. A new program, Lipo, was developed that can analyse entire predicted proteomes and give a list of recognised lipoproteins categorised according to their lipo-box similarity to known Gram-negative lipoproteins ( http://www.bioinfo.no/tools/lipo ). This report is the first using a proteomics and bioinformatics approach to identify the OM subproteome of an obligate methanotroph.