U6 Promoter Research Articles

Genome editing of WSSV CRISPR/Cas9 and immune activation extends the survival of infected Penaeus vannamei

White spot syndrome virus (WSSV) is an exceptionally harmful virus that generally causes high levels of mortality in cultured shrimp. Attempts at viral suppression have been made to control the disease and have achieved limited efficiency. Recent advances in genome editing technology using CRISPR/Cas9 have led to potential innovations to prevent or treat many viral diseases. In this study, a CRISPR/Cas9 system was applied to WSSV genome cleavage to suppress WSSV infection in shrimp. The U6 promoter sequence was identified. A chimeric DNA vector consisting of the shrimp U6 promoter with gRNA expression sequences specific to two sites of the WSSV genome and the WSSV ribonucleotide reductase promoter with the Cas9 DNA sequence in pAC-sgRNA-Cas9 was constructed. The expression of gRNAs specific to the WSSV genome and Cas9 was determined in primary cultured hemocyte cells and in shrimp tissue via RT‒PCR. The efficacy of CRISPR/Cas9-WSSV for WSSV genome cleavage was determined in vitro and against WSSV-infected Penaeus vannamei. The reaction of synthetic gRNAs and recombinant Cas9 was able to cleave WSSV DNA amplicons, and shrimp that received CRISPR/Cas9-WSSV presented significantly lower WSSV DNA. In addition to interfering with viral DNA propagation, CRISPR/Cas9-WSSV encapsulated with IHHNV-VLP also stimulated an immune-related gene response. Treatment with CRISPR/Cas9-WSSV against WSSV challenge resulted in a significantly longer survival period. This finding has led to the development and application of a CRISPR/Cas9 system for WSSV infectious disease control, which could be used for managing shrimp aquaculture in the future.

Design and construction of a GAP-43 reporter system for potential identification of effective therapeutics for peripheral nerve regeneration

Background: Peripheral nerve injury (PNI) is a condition that can result in muscle paralysis and sensory disturbances. Electrical stimulation and/or the application of exogenous neurotrophic factors and cytokines are effective at enhancing nerve regeneration and is mediated via the expression of regeneration associated genes (RAGs) such as the growth associated protein GAP-43. Therapeutic upregulation of GAP-43 has potential use as a treatment for improving recovery from PNI. Few studies have investigated the potential of increasing GAP-43 for PNI therapeutic purposes and current methods for measuring GAP-43 expression are limited. Aims and Objectives: The broader aim of this work was to construct a motor neuron-like cell model with a GAP-43 reporter system. Such a model would have potential use in screening for novel therapeutics that upregulate GAP-43 and in the optimisation of electrical stimulation treatment in combination with these therapies. The key aim of the work was to design and construct a Cas9 expressing plasmid bearing a gRNA that targets GAP-43 cleavage and a donor plasmid bearing a reporter GFP or Neo cassette flanked with 5’ and 3’ GAP-43 homology arms (HAs) to facilitate the insertion of the reporter immediately 3’ of the GAP-43 promoter via CRISPR/Cas9 homology directed repair (HDR). Such an insertion would enable quantitative measurement of endogenous expression of the GAP-43 gene. Methods and Results: To guide the Cas9 nuclease to the target location, GAP-43 gRNA oligomers were designed and cloned downstream of the U6 promoter in the Cas9 expression plasmid px330, which also expresses the Cas9 gene and the cloned gRNA when transfected into cells. For CRISPR/Cas9 HDR, the 5’ and 3’ GAP-43 HAs were amplified from mouse genomic DNA and cloned into the donor plasmid using Gibson Assembly so that they flanked the reporter cassette. Results: This work successfully constructed the Cas9 gRNA expressing plasmid to target cleavage of the GAP-43 gene in mouse cell lines and has provided the complete design and construction foundation for generation of the reporter system for the endogenous GAP-43 gene in mice.

Utility of Arabidopsis KASII Promoter in Development of an Effective CRISPR/Cas9 System for Soybean Genome Editing and Its Application in Engineering of Soybean Seeds Producing Super-High Oleic and Low Saturated Oils.

This study reports the use of the Arabidopsis KASII promoter (AtKASII) to develop an efficient CRISPR/Cas9 system for soybean genome editing. When this promoter was paired with Arabidopsis U6 promoters to drive Cas9 and single guide RNA expression, respectively, simultaneous editing of the three fatty acid desaturase genes GmFAD2-1A, GmFAD2-1B, and GmFAD3A occurred in more than 60% of transgenic soybean lines at T2 generation, and all the triple mutants possessed desirable high-oleic traits. In sharp contrast, not a single line underwent simultaneous editing of the three target genes when AtKASII was replaced by the widely used AtEC1.2 promoter. Furthermore, our study showed that the stable and inheritable mutations in the high-oleic lines did not alter the overall contents of oil and protein or amino acid composition while increasing the oleic acid content up to 87.6% from approximately 23.8% for wild-type seeds, concomitant with 34.4- and 3.7-fold reductions in linoleic and linolenic acid, respectively. Collectively, this study demonstrates that the AtKASII promoter is highly promising for optimization of the CRISPR/Cas9 system for genome editing in soybean and possibly beyond.

CRISPR/Cas9-based genome engineering in the filamentous fungus Rhizopus oryzae and its application to L-lactic acid production.

The filamentous fungus Rhizopus oryzae is one of the main industrial strains for the production of a series of important chemicals such as ethanol, lactic acid, and fumaric acid. However, the lack of efficient gene editing tools suitable for R. oryzae makes it difficult to apply technical methods such as metabolic engineering regulation and synthetic biology modification. A CRISPR-Cas9 system suitable for efficient genome editing in R. oryzae was developed. Firstly, four endogenous U6 promoters of R. oryzae were identified and screened with the highest transcriptional activity for application to sgRNA transcription. It was then determined that the U6 promoter mediated CRISPR/Cas9 system has the ability to efficiently edit the genome of R. oryzae through NHEJ and HDR-mediated events. Furthermore, the newly constructed CRISPR-Cas9 dual sgRNAs system can simultaneously disrupt or insert different fragments of the R. oryzae genome. Finally, this CRISPR-Cas9 system was applied to the genome editing of R. oryzae by knocking out pyruvate carboxylase gene (PYC) and pyruvate decarboxylase gene (pdcA) and knocking in phosphofructokinase (pfkB) from Escherichia coli and L-lactate dehydrogenase (L-LDH) from Heyndrickxia coagulans, which resulted in a substantial increase in L-LA production. In summary, this study showed that the CRISPR/Cas9-based genome editing tool is efficient for manipulating genes in R. oryzae.

Exploring the Use of Alternative Promoters for Enhanced Transgene and sgRNA Expression in Atlantic Salmon Cells.

This study facilitates design of expression vectors and lentivirus tools for gene editing of Atlantic salmon. We have characterized widely used heterologous promoters and novel endogenous promoters in Atlantic salmon cells. We used qPCR to evaluate the activity of several U6 promoters for sgRNA expression, including human U6 (hU6), tilapia U6 (tU6), mouse U6 (mU6), zebrafish U6 (zU6), Atlantic salmon U6 (sU6), medaka U6 (medU6), and fugu U6 (fU6) promoters. We also evaluated several polymerase type II (pol II) promoters by luciferase assay. Our results showed that hU6 and tU6 promoters were the most active among all the tested U6 promoters, and heterologous promoters (CMV, hEF1α core) had higher activity compared to endogenous Atlantic salmon promoters sHSP8, sNUC3L, sEF1α. Among endogenous pol II promoters, sEF1α and sHSP8 displayed higher activity than sNUC3L, sHSP703, sHSP7C, sXRCC1L, and sETF. We observed that extending the promoter sequence to include the region up to the start codon (ATG) resulted in a significant increase in expression efficiency for sNUC3L and sEF1α. We also show that mutating the PRDM1 motif will significantly decrease the activity of the sEF1α promoter. The presence of the PRDM1 motif in sHSP8 promoter was also associated with relatively high expression compared to the promoters that naturally lacked this motif, such as sNUC3L. We speculate that this short sequence might be included in other promoters to further enhance the promoter activity, but further experiments are needed to confirm this. Our findings provide valuable insights into the activity of different promoters in Atlantic salmon cells and can be used to facilitate further transgenic studies and improve the efficiency of transgene expression in Atlantic salmon.

An optimised CRISPR Cas9 and Cas12a mutagenesis toolkit for Barley and Wheat

BackgroundCRISPR Cas9 and Cas12a are the two most frequently used programmable nucleases reported in plant systems. There is now a wide range of component parts for both which likely have varying degrees of effectiveness and potentially applicability to different species. Our aim was to develop and optimise Cas9 and Cas12a based systems for highly efficient genome editing in the monocotyledons barley and wheat and produce a user-friendly toolbox facilitating simplex and multiplex editing in the cereal community.ResultsWe identified a Zea mays codon optimised Cas9 with 13 introns in conjunction with arrayed guides driven by U6 and U3 promoters as the best performer in barley where 100% of T0 plants were simultaneously edited in all three target genes. When this system was used in wheat > 90% of T0 plants were edited in all three subgenome targets. For Cas12a, an Arabidopsis codon optimised sequence with 8 introns gave the best editing efficiency in barley when combined with a tRNA based multiguide array, resulting in 90% mutant alleles in three simultaneously targeted genes. When we applied this Cas12a system in wheat 86% & 93% of T0 plants were mutated in two genes simultaneously targeted. We show that not all introns contribute equally to enhanced mutagenesis when inserted into a Cas12a coding sequence and that there is rationale for including multiple introns. We also show that the combined effect of two features which boost Cas12a mutagenesis efficiency (D156R mutation and introns) is more than the sum of the features applied separately.ConclusionBased on the results of our testing, we describe and provide a GoldenGate modular cloning system for Cas9 and Cas12a use in barley and wheat. Proven Cas nuclease and guide expression cassette options found in the toolkit will facilitate highly efficient simplex and multiplex mutagenesis in both species. We incorporate GRF-GIF transformation boosting cassettes in wheat options to maximise workflow efficiency.

Construction and Stability of All-in-One Adenovirus Vectors Simultaneously Expressing Four and Eight Multiplex Guide RNAs and Cas9 Nickase.

CRISPR/Cas9 technology is expected to offer novel genome editing-related therapies for various diseases. We previously showed that an adenovirus vector (AdV) possessing eight expression units of multiplex guide RNAs (gRNAs) was obtained with no deletion of these units. Here, we attempted to construct "all-in-one" AdVs possessing expression units of four and eight gRNAs with Cas9 nickase, although we expected obstacles to obtain complete all-in-one AdVs. The first expected obstacle was that extremely high copies of viral genomes during replication may cause severe off-target cleavages of host cells and induce homologous recombination. However, surprisingly, four units in the all-in-one AdV genome were maintained completely intact. Second, for the all-in-one AdV containing eight gRNA units, we enlarged the E3 deletion in the vector backbone and shortened the U6 promoter of the gRNA expression units to shorten the AdV genome within the adenovirus packaging limits. The final size of the all-in-one AdV genome containing eight gRNA units still slightly exceeded the reported upper limit. Nevertheless, approximately one-third of the eight units remained intact, even upon preparation for in vivo experiments. Third, the genome editing efficiency unexpectedly decreased upon enlarging the E3 deletion. Our results suggested that complete all-in-one AdVs containing four gRNA units could be obtained if the problem of the low genome editing efficiency is solved, and those containing even eight gRNA units could be obtained if the obstacle of the vector size is also removed.

Design and evaluation of antisense sequence length for modified mouse U7 small nuclear RNA to induce efficient pre-messenger RNA splicing modulation in vitro.

Pre-messenger RNA (pre-mRNA) splicing modulation is an attractive approach for investigating the mechanisms of genetic disorders caused by mis-splicing. Previous reports have indicated that a modified U7 small nuclear RNA (U7 snRNA) is a prospective tool for modulating splicing both in vitro and in vivo. To date, very few studies have investigated the role of antisense sequence length in modified U7 snRNA. In this study, we designed a series of antisense sequences with various lengths and evaluated their efficiency in inducing splicing modulation. To express modified U7 snRNAs, we constructed a series of plasmid DNA sequences which codes cytomegalovirus (CMV) enhancer, human U1 promoter, and modified mouse U7 snRNAs with antisense sequences of different lengths. We evaluated in vitro splicing modulation efficiency using a luciferase reporter system for simple and precise evaluation as well as reverse transcription-polymerase chain reaction to monitor splicing patterns. Our in vitro assay findings suggest that antisense sequences of modified mouse U7 snRNAs have an optimal length for efficient splicing modulation, which depends on the target exon. In addition, antisense sequences that were either too long or too short decreased splicing modulation efficiency. To confirm reproducibility, we performed an in vitro assay using two target genes, mouse Fas and mouse Dmd. Together, our data suggests that the antisense sequence length should be optimized for modified mouse U7 snRNAs to induce efficient splicing modulation.

Identification of active shrimp U6 promoters for efficient CRISPR/Cas9 gene editing in penaeid shrimps based on a VP28-pseudotyped baculovirus expression system

The activity of species-specific U6 promoter is crucial for efficient expression of sgRNA in target cells when CRISPR/Cas9 is delivered in plasmid, thus the use of CRISPR/Cas9 technology was markedly limited in shrimps due to the lack of endogenous U6 promoter. To overcome this, in this study four candidate U6 promoters (LvU6 A-D) had been identified and cloned from the genome of penaeid shrimp (Litopenaeus vannamei), and then their activities to drive the expression of shRNA in insect Sf9 cells and shrimp primary hemolymph cells were successively analyzed using insect baculovirus expression system. It was found that all the isolated four LvU6 promoters contained all the characteristic elements (DSE, PSE and TATA box) of RNA polymerase III U6 promoter. Both a dual plasmids delivery system (Bacmid-PH-LvU6-shEGFP-P2-gBFP and Bacmid-PH-EGFP) and an all-in-one plasmid delivery system (Bacmid-PH-EGFP-LvU6-shEGFP-P2-gBFP) were constructed for RNAi analysis of reporter gene EGFP which are not pseudo-typed for the infection of Sf9 cells but pseudo-typed for the infection of shrimp cells by co-transfection with pFastBac1-VP28 (an envelope protein of shrimp virus). The obtained results showed that only LvU6 A, B and D could successfully drive the expression of shEGFP in both of the tested cells, and the order of interference efficiency was LvU6 B > LvU6 D > LvU6 A. Furthermore, the RNAi efficiency of LvU6 B was up to 27% (P < 0.05) when the dual plasmids were co-transfected at a mass ratio of 1: 0.1 in Sf9 cells, which was higher than that of the all-in-one plasmid (15%, P < 0.05). In shrimp cells, no noticeable fluorescence could be observed when dual plasmids delivery system was used; in contrast, the efficiency could reach 19% when all-in-one delivery system was used (P < 0.05). However, when LvU6 B was used in the shrimp CRISPR/Cas9 gene editing system with a co-expressed VP28 gene in the baculovirus vector, there were no mutations detected in the target gene (Bax) of both in vitro cultured shrimp cells and adult shrimps. Surprisingly, after optimizing the system by using a truncated LvU6 B promoter and introducing double sgRNAs, the insertion, deletion or replacement of target sites occurred in both shrimp cells and adult tissues (spermatophore, Oka and intestine), and the mutation rates increased up to 22% and 46%, respectively. This work has provided one active shrimp U6 promoter for efficient gene editing in shrimps and shrimp cells.

Development of a Csy4-Processing TRV-Based CRISPR/Cas9 Genome Editing System in Nicotiana benthamiana

AbstractThe CRISPR/Cas9 system is a site-specific genome editing tool that has been widely used in various plant species. The plant virus-based gRNA (guide RNA) delivery system, which differs from the typical Agrobacterium-mediated transformation, is an attractive method to facilitate the application of CRISPR/Cas9. The virally delivered gRNA is usually driven by heterologous plant U6 or viral promoters (e.g., pea early-browning virus, PEBV; barley stripe mosaic virus, BSMV). However, heterologous promoters may have poor performance in some cases. In this paper, a feasible option to detach gRNA(s) from the virus genome is employed. Specifically, the Csy4-RNA processing system is used to release gRNA(s) from the tobacco rattle virus (TRV). The coding sequences of Cas9 and Csy4 nucleases are cloned into a single polycistronic expression cassette under an estrogen-inducible promoter in a binary vector, and the gRNA is cloned into the TRV genome flanked by two 20 bp Csy4 recognition sites. The results show that the Csy4-processing TRV-based delivery system works effectively in targeting single and multiple sites, nucleotide replacement, and large fragment deletion in Cas9-mediated genome editing via transient expression in Nicotiana benthamiana. The Csy4-TRV is a promising gRNA(s) processing and delivery system for CRISPR/Cas9 genome editing. This method can be easily adapted to other plant RNA viruses, facilitating the application of the CRISPR/Cas9 system in plants.

An Efficient Vector-Based CRISPR/Cas9 System in Zebrafish Cell Line.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been widely applied in animals as an efficient genome editing tool. However, the technique is difficult to implement in fish cell lines partially due to the lack of efficient promoters to drive the expression of both sgRNA and the Cas9 protein within a single vector. In this study, it was indicated that the zebrafish U6 RNA polymerase III (ZFU6) promoter could efficiently induce tyrosinase (tyr) gene editing and lead to loss of retinal pigments when co-injection with Cas9 mRNA in zebrafish embryo. Furthermore, an optimized all-in-one vector for expression of the CRISPR/Cas9 system in the zebrafish fibroblast cell line (PAC2) was constructed by replacing the human U6 promoter with ZFU6 promoter, basing on the lentiCRISPRV2 system that widely applied in mammal cells. This new vector could successfully target the cellular communication network factor 2a (ctgfa) gene and demonstrated its function in the PAC2 cell. Notably, the vector could also be used to edit the endogenous EMX1 gene in the mammal 293T cell line, implying its wide application potential. In conclusion, we established a new gene editing tool for zebrafish cell line, which could be a useful in vitro platform for high-throughput analyzing gene function in fish.

Validation of endogenous U6 promoters for expanding the CRISPR toolbox in Nicotiana tabacum

Validation of endogenous U6 promoters for expanding the CRISPR toolbox in Nicotiana tabacum

Targeting thrombospondin-2 retards liver fibrosis by inhibiting TLR4-FAK/TGF-β signaling

Thrombospondin-2 (THBS2) expression is associated with liver fibrosis regardless of etiology. However, the role of THBS2 in the pathogenesis of liver fibrosis has yet to be elucidated. The invivo effects of silencing Thbs2 in hepatic stellate cells (HSCs) were examined using an adeno-associated virus vector (serotype 6, AAV6) containing short-hairpin RNAs targeting Thbs2, under the regulatory control of cytomegalovirus, U6 or the α-smooth muscle promoter, in mouse models of carbon tetrachloride or methionine-choline deficient (MCD) diet-induced liver fibrosis. Crosstalk between THBS2 and toll-like receptor 4 (TLR4), as well as the cascaded signaling, was systematically investigated using mouse models, primary HSCs, and human HSC cell lines. THBS2 was predominantly expressed in activated HSCs and dynamically increased with liver fibrosis progression and decreased with regression. Selective interference of Thbs2 in HSCs retarded intrahepatic inflammatory infiltration, steatosis accumulation, and fibrosis progression following carbon tetrachloride challenge or in a dietary model of metabolic dysfunction-associated steatohepatitis. Mechanically, extracellular THBS2, as a dimer, specifically recognized and directly bound to TLR4, activating HSCs by stimulating downstream profibrotic focal adhesion kinase (FAK)/transforming growth factor beta (TGF-β) pathways. Disruption of the THBS2-TLR4-FAK/TGF-β signaling axis notably alleviated HSC activation and liver fibrosis aggravation. THBS2 plays a crucial role in HSC activation and liver fibrosis progression through TLR4-FAK/TGF-β signaling in an autocrine manner, representing an attractive potential therapeutic target for liver fibrosis. Thrombospondin-2 (THBS2) is emerging as a factor closely associated with liver fibrosis regardless of etiology. However, the mechanisms by which THBS2 is involved in liver fibrosis remain unclear. Here, we showed that THBS2 plays a prominent role in the pathogenesis of liver fibrosis by activating the TLR4-TGF-β/FAK signaling axis and hepatic stellate cells in an autocrine manner, providing a potential therapeutic target for the treatment of liver fibrosis.

Multiplexed transcriptomic profiling of the fate of human CAR T cells in vivo via genetic barcoding with shielded small nucleotides.

The design of chimeric antigen receptor (CAR) T cells would benefit from knowledge of the fate of the cells in vivo. This requires the permanent labelling of CAR T cell products and their pooling in the same microenvironment. Here, we report a cell-barcoding method for the multiplexed longitudinal profiling of cells in vivo using single-cell RNA sequencing (scRNA-seq). The method, which we named shielded-small-nucleotide-based scRNA-seq (SSN-seq), is compatible with both 3' and 5' single-cell profiling, and enables the recording of cell identity, from cell infusion to isolation, by leveraging the ubiquitous Pol III U6 promoters to robustly express small-RNA barcodes modified with direct-capture sequences. By using SSN-seq to track the dynamics of the states of CAR T cells in a tumour-rechallenge mouse model of leukaemia, we found that a combination of cytokines and small-molecule inhibitors that are used in the ex vivo manufacturing of CAR T cells promotes the in vivo expansion of persistent populations of CD4+ memory T cells. By facilitating the probing of cell-state dynamics in vivo, SSN-seq may aid the development of adoptive cell therapies.

Development of a CRISPR/Cpf1 system for multiplex gene editing in Aspergillus oryzae.

CRISPR/Cas technology is a powerful tool for genome engineering in Aspergillus oryzae as an industrially important filamentous fungus. Previous study has reported the application of the CRISPR/Cpf1 system based on the Cpf1 (LbCpf1) from Lachnospiraceae bacterium in A. oryzae. However, multiplex gene editing have not been investigated using this system. Here, we presented a new CRISPR/Cpf1 multiplex gene editing system in A. oryzae, which contains the Cpf1 nuclease (FnCpf1) from Francisella tularensis subsp. novicida U112 and CRISPR-RNA expression cassette. The crRNA cassette consisted of direct repeats and guide sequences driven by the A. oryzae U6 promoter and U6 terminator. Using the constructed FnCpf1 gene editing system, the wA and pyrG genes were mutated successfully. Furthermore, simultaneous editing of wA and pyrG genes in A. oryzae was performed using two guide sequences targeting these gene loci in a single crRNA array. This promising CRISPR/Cpf1 genome-editing system provides a powerful tool for genetically engineering A. oryzae.

CRISPR/Cas9 editing characteristics of multiple transgenic generations in Fortunella hindsii, an early flowering mini-citrus

The CRISPR/Cas9 editing system is presently regarded as one of the most efficient tools for genome editing and has been implemented in perennial fruit crops. However, the characteristics of transgenic plants and their subsequent generation have rarely been reported in fruit trees. In this study, 15 distinct preserved germplasm resources of Hongkong kumquat (Fortunella hindsii) were utilized to selecting suitable genotypes for genetic transformation. The shoot regeneration efficiency, seed number per fruit, and epicotyl diameter displaid a moderate positive correlation with transformation efficiency, with correlation coefficients of 0.6267, 0.5429, and 0.5388, respectively. The transcription factor RKD family gene FhRWP was knocked out in kumquat, resulting in the production of seven T0 and 34 T1 plants. Editing efficiency of T0 generation ranged from 1.62% to 68.11%, with a significant difference in editing activity among cases. Up to 23 editing types were detected in a single positive transformant, demonstrating that the high degree of citrus editing chimerism. Not all of T1 lines exhibited an increase in editing efficiency relative to T0. The descendants of T0-A2/A3 were all negative, while the other group of T1 lines showed both higher chimera and editing efficiencies of up to 92% than the others. Finally, the CRISPR/Cas9 vector was improved by using the endogenous U6 promoter in F. hindsii. This study provides novel insights into the application of the CRISPR/Cas9 system in asexual plants and serves as a reference for gene function verification in citrus and other species.

Cloning and Functional Verification of Endogenous U6 Promoters for the Establishment of Efficient CRISPR/Cas9-Based Genome Editing in Castor (Ricinus communis).

Castor (Ricinus communis) seeds are rich in a type of hydroxy fatty acid called ricinoleic acid, which is in high demand for the production of plant-based plastics, lubricants, and hydraulic oils. However, the high content of ricin, a toxic protein, in these seeds has restricted further expansion in the area of castor cultivation. Therefore, the development of ricin-free castor is needed. Genome editing technology, although successfully applied in several plant species, is still in the developing stages in castor and awaits the identification of an endogenous U6 promoter with robust function. Here, we searched for U6 small nuclear RNA (snRNA) genes in the castor genome. This led to the identification of six U6 snRNA genes. The promoters of these U6 snRNA genes were cloned, and their function was examined in castor cells using the particle delivery method. The results showed that a U6 promoter length of approximately 300 bp from the transcription start site was sufficient to activate gene expression. This study provides insights into the endogenous castor U6 promoter sequences and outlines a method for verifying the function of U6 promoters in plants using the particle delivery system.

Cloning and expression analysis of U6 promoters in Panax quinquefolius

The U6 promoter is an important element driving sgRNA transcription in the CRISPR/Cas9 system. Seven PqU6 promo-ter sequences were cloned from the gDNA of Panax quinquefolium, and the transcriptional activation ability of the seven promoters was studied. In this study, seven PqU6 promoter sequences with a length of about 1 300 bp were cloned from the adventitious roots of P. quinquefolium cultivated for 5 weeks. Bioinformatics tools were used to analyze the sequence characteristics of PqU6 promoters, and the fusion expression vectors of GUS gene driven by PqU6-P were constructed. Tobacco leaves were transformed by Agrobacterium tumefaciens-mediated method for activity detection. The seven PqU6 promoters were truncated from the 5'-end to reach 283, 287, 279, 289, 295, 289, and 283 bp, respectively. The vectors for detection of promoter activity were constructed with GUS as a reported gene and used to transform P. quinquefolium callus and tobacco leaves. The results showed that seven PqU6 promoter sequences(PqU6-1P to PqU6-7P) were cloned from the gDNA of P. quinquefolium, with the length ranged from 1 246 bp to 1 308 bp. Sequence comparison results showed that the seven PqU6 promoter sequences and the AtU6-P promoter all had USE and TATA boxes, which are essential elements affecting the transcriptional activity of the U6 promoter. The results of GUS staining and enzyme activity test showed that all the seven PqU6 promoters had transcriptional activity. The PqU6-7P with a length of 1 269 bp had the highest transcriptional activity, 1.31 times that of the positive control P-35S. When the seven PqU6 promoters were truncated from the 5'-end(PqU6-1PA to PqU6-7PA), their transcriptional activities were different in tobacco leaves and P. quinquefolium callus. The transcriptional activity of PqU6-7PA promoter(283 bp) was 1.59 times that of AtU6-P promoter(292 bp) when the recipient material was P. quinquefolium callus. The findings provide more ideal endogenous U6 promoters for CRISPR/Cas9 technology in ginseng and other medicinal plants.

Molecular Characterization of U6 Promoters from Orange-Spotted Grouper (Epinephelus coioides) and Its Application in DNA Vector-Based RNAi Technology.

The U6 promoter, a typical RNA polymerase III promoter, is widely used to transcribe small RNAs in vector-based siRNA systems. The RNAi efficiency is mainly dependent on the transcriptional activity of the U6 promoter. However, studies have found that U6 promoters isolated from some fishes do not work well in distantly related species. To isolate a U6 promoter with high transcriptional efficiency from fish, in this study, we cloned five U6 promoters in orange-spotted grouper, of which only the grouper U6-1 (GU6-1) promoter contains the OCT element in the distant region. Functional studies revealed that the GU6-1 promoter has high transcriptional ability, which could efficiently transcribe shRNA and result in target gene knockdown in vitro and in vivo. Subsequently, the deletion or mutation of the OCT motif resulted in a significant decrease in promoter transcriptional activity, demonstrating that the OCT element plays an important role in enhancing the grouper U6 promoter transcription. Moreover, the transcriptional activity of the GU6-1 promoter showed little species specificity. It not only works in the grouper but also possesses high transcriptional activity in the zebrafish. Knockdown of the mstn gene in zebrafish and grouper through shRNA driven by the GU6-1 promoter could promote fish growth, suggesting that the GU6-1 promoter can be used as a potential molecular tool in aquaculture practice.

U3 snoRNA-mediated degradation of ZBTB7A regulates aerobic glycolysis in isocitrate dehydrogenase 1 wild-type glioblastoma cells.

The isocitrate dehydrogenase (IDH) phenotype is associated with reprogrammed energy metabolism in glioblastoma (GBM) cells. Small nucleolar RNAs (snoRNAs) are known to exert an important regulatory role in the energy metabolism of tumor cells. The purpose of this study was to investigate the role of C/D box snoRNA U3 and transcription factor zinc finger and BTB domain-containing 7A (ZBTB7A) in the regulation of aerobic glycolysis and the proliferative capacity of IDH1 wild-type (IDH1WT ) GBM cells. Quantitative reverse transcription PCR and western blot assays were utilized to detect snoRNA U3 and ZBTB7A expression. U3 promoter methylation status was analyzed via bisulfite sequencing and methylation-specific PCR. Seahorse XF glycolysis stress assays, lactate production and glucose consumption measurement assays, and cell viability assays were utilized to detect glycolysis and proliferation of IDH1WT GBM cells. We found that hypomethylation of the CpG island in the promoter region of U3 led to the upregulation of U3 expression in IDH1WT GBM cells, and the knockdown of U3 suppressed aerobic glycolysis and the proliferation ability of IDH1WT GBM cells. We found that small nucleolar-derived RNA (sdRNA) U3-miR, a small fragment produced by U3, was able to bind to the ZBTB4 3'UTR region and reduce ZBTB7A mRNA stability, thereby downregulating ZBTB7A protein expression. Furthermore, ZBTB7A transcriptionally inhibited the expression of hexokinase 2 (HK2) and lactate dehydrogenase A (LDHA), which are key enzymes of aerobic glycolysis, by directly binding to the HK2 and LDHA promoter regions, thereby forming the U3/ZBTB7A/HK2 LDHA pathway that regulates aerobic glycolysis and proliferation of IDH1WT GBM cells. U3 enhances aerobic glycolysis and proliferation in IDH1WT GBM cells via the U3/ZBTB7A/HK2 LDHA axis.