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.

Identification and functional analysis of Cochliomyia hominivorax U6 gene promoters.

The New World screwworm, Cochliomyia hominivorax, is an obligate parasite, which is a major pest of livestock. While the sterile insect technique was used very successfully to eradicate C. hominivorax from North and Central America, more cost-effective genetic methods will likely be needed in South America. The recent development of CRISPR/Cas9-based genetic approaches, such as homing gene drive, could provide a very efficient means for the suppression of C. hominivorax populations. One component of a drive system is the guide RNA(s) driven by a U6 gene promoter. Here, we have developed an in vivo assay to evaluate the activity of the promoters from seven C. hominivorax U6 genes. Embryos from the related blowfly Lucilia cuprina were injected with plasmid DNA containing a U6-promoter-guide RNA construct and a source of Cas9, either protein or plasmid DNA. Activity was assessed by the number of site-specific mutations in the targeted gene in hatched larvae. One promoter, Chom U6_b, showed the highest activity. These U6 gene promoters could be used to build CRISPR/Cas9-based genetic systems for the control of C. hominivorax.

[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.

Efficient genome editing using endogenous U6 snRNA promoter-driven CRISPR/Cas9 sgRNA in Sclerotinia sclerotiorum.

We previously reported on a CRISPR-Cas9 genome editing system for the necrotrophic fungal plant pathogen Sclerotinia sclerotiorum. This system (the TrpC-sgRNA system), based on an RNA polymerase II (RNA Pol II) promoter (TrpC) to drive sgRNA transcription in vivo, was successful in creating gene insertion mutants. However, relatively low efficiency targeted gene editing hampered the application of this method for functional genomic research in S. sclerotiorum. To further optimize the CRISPR-Cas9 system, a plasmid-free Cas9 protein/sgRNA ribonucleoprotein (RNP)-mediated system (the RNP system) and a plasmid-based RNA polymerase III promoter (U6)-driven sgRNA transcription system (the U6-sgRNA system) were established and evaluated. The previously characterized oxaloacetate acetylhydrolase (Ssoah1) locus and a new locus encoding polyketide synthase12 (Sspks12) were targeted in this study to create loss-of-function mutants. The RNP system, similar to the TrpC-sgRNA system we previously reported, creates mutations at the Ssoah1 gene locus with comparable efficiency. However, neither system successfully generated mutations at the Sspks12 gene locus. The U6-sgRNA system exhibited a significantly higher efficiency of genemutation at both loci. This technology provides a simple and efficient strategy for targeted gene mutation and thereby will accelerating the pace of research of pathogenicity and development in this economically important plant pathogen.

Genome editing efficiency of four Drosophila suzukii endogenous U6 promoters.

The invasive spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) has caused serious economic losses to the fruit industry. The conventional control methods have many limitations and genetic engineering technologies such as CRISPR/Cas9-mediated gene drive are promising approaches. In the CRISPR/Cas9 system, the transcriptional regulatory elements play an important role in the activities of gRNA. Thus, in order to improve the genome editing efficiency of the CRISPR/Cas9 system in D. suzukii, we cloned and tested four endogenous U6 promoters to drive mutagenesis of the white gene. Our results showed that all the four promoters could be used with variable efficiency. The promoter DsU6-3 had the highest genome editing efficiency among the four DsU6 promoters. Compared with the DsU6-3 promoter, the DmU6:3 promoter showed lower efficiency to drive mutagenesis in D. suzukii. These findings expand the range of promoters available to express gRNAs in D. suzukii, facilitating the basic and applied research on this important pest.

A novel vector-based RNAi method using mouse U6 promoter-driven shRNA expression in the filamentous fungus Blakeslea trispora.

PURPOSE: There are several studies on the use of RNA interference (RNAi) for gene function analysis in fungi. However, most studies on filamentous fungi are based on in vitro-transcribed or -synthesized small interfering RNA (siRNA), and only a few have reported the use of vector-based RNAi. Here we want to develop and evaluate a new vector-based RNAi method using the mouse U6 promoter to drive short hairpin RNA (shRNA) expression in the filamentous fungi. METHODS: Molecular techniques were employed to develop and evaluate a new vector-based RNAi method using the mouse U6 promoter to drive short hairpin RNA (shRNA) expression in the filamentous fungus Blakeslea trispora. RESULTS: We characterized the mouse U6 promoter and utilized it for the expression of shRNA in B. trispora. Using real-time polymerase chain reaction and western blotting analyses, we confirmed the decrease in the mRNA and protein expression of carRA, respectively, in cells transformed with the mouse U6 promoter-driven shRNA expression vector. This indicated that the shRNA was transcribed from the mouse U6 promoter and correctly processed into siRNA and that the mouse U6 promoter exhibited transcription ability in the filamentous fungi. CONCLUSIONS: The results suggest that the mouse U6 promoter that drives the expression of shRNA vectors may serve as a novel tool for RNAi induction in filamentous fungi.

Enhancing the CRISPR/Cas9 system based on multiple GmU6 promoters in soybean.

Small guide RNA (sgRNA) is an important component of the CRISPR/Cas9 system. The gene editing efficiency of the CRISPR/Cas9 system could be enhanced by using highly active U6 promoters to drive the expression of sgRNA. Therefore, we constructed various expression vectors based on the 11 GmU6 promoters predicted and cloned in the whole soybean genome. The expression of truncated GUS driven by 11 GmU6 promoters was tested in hairy roots and by Arabidopsis thaliana transformation. The results indicated that higher transcriptional levels were driven by 5 GmU6 promoters (GmU6-4, GmU6-7, GmU6-8, GmU6-10 and GmU6-11) in both soybean hairy roots and Arabidopsis thaliana. In addition, three genes, Glyma03g36470, Glyma14g04180 and Glyma06g136900, were selected as targets to detect the transcriptional levels of multiple GmU6 promoters. Mutations in these three genes were detected in soybean hairy roots after Agrobacterium rhizogenes infection, indicating efficient target gene editing, including nucleotide insertion, deletion, and substitution. Mutation efficiencies differed among the 11 GmU6 promoters, ranging from 2.8% to 20.6%, and markedly higher efficiencies were obtained with all three genes using the GmU6-8 (20.3%) and GmU6-10 (20.6%) promoters. These two GmU6 promoters also showed higher ability to drive truncated GUS transcription in both soybean hairy roots and transformed Arabidopsis thaliana. These results will help to construct an efficient CRISPR-Cas9 gene editing system and promote the application of the CRISPR-Cas9 genome editing system in soybean molecular breeding.

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 293 T 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.

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.

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.

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.

Establishment of highly efficient transgenic system for black soldier fly (Hermetia illucens).

The black soldier fly (BSF), Hermetia illucens, is a promising insect for mitigating solid waste problems as its larvae are able to bioconvert organic waste into valuable biomass. We recently reported a high-quality genome assembly of the BSF; analysis of this genome sequence will further the understanding of insect biology and identify genes that can be manipulated to improve efficiency of bioconversion. To enable genetic manipulation of the BSF, we have established the first transgenic methods for this economically important insect. We cloned and identified the ubiquitous actin5C promoter (Hiactin5C-p3k) and 3 endogenous U6 promoters (HiU6:1, HiU6:2, and HiU6:3). The Hiactin5C promoter was used to drive expression of a hyperactive variant of the piggyBac transposase, which exhibited up to 6-fold improvement in transformation rate when compared to the wild-type transposase. Furthermore, we evaluated the 3 HiU6 promoters using this transgenic system. HiU6:1 and HiU6:2 promoters provided the highest knockdown efficiency with RNAi and are thus promising candidates for future Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) development. Overall, our findings provide valuable genetic engineering toolkits for basic research and genetic manipulation of the BSF.

Establishment of an Integrated CRISPR/Cas9 Plasmid System for Simple and Efficient Genome Editing in Medaka In Vitro and In Vivo.

Although CRISPR/Cas9 has been used in gene manipulation of several fish species in vivo, its application in fish cultured cells is still challenged and limited. In this study, we established an integrated CRISPR/Cas9 plasmid system and evaluated its efficiency of gene knock-out or knock-in at a specific site in medaka (Oryzias latipes) in vitro and in vivo. By using the enhanced green fluorescent protein reporter plasmid pGNtsf1, we demonstrate that pCas9-U6sgRNA driven by endogenous U6 promoter (pCas9-mU6sgRNA) mediated very high gene editing efficiency in medaka cultured cells, but not by exogenous U6 promoters. After optimizing the conditions, the gene editing efficiencies of eight sites targeting for four endogenous genes were calculated, and the highest was up to 94% with no detectable off-target. By one-cell embryo microinjection, pCas9-mU6sgRNA also mediated efficient gene knock-out in vivo. Furthermore, pCas9-mU6sgRNA efficiently mediated gene knock-in at a specific site in medaka cultured cells as well as embryos. Collectively, our study demonstrates that the genetic relationship of U6 promoter is critical to gene editing efficiency in medaka cultured cells, and a simple and efficient system for medaka genome editing in vitro and in vivo has been established. This study provides an insight into other fish genome editing and promotes gene functional analysis.

Enhancement of the CRISPR/Cas9-Based Genome Editing System in Lettuce (Lactuca sativa L.) Using the Endogenous U6 Promoter.

The CRISPR/Cas9 system has been widely applied as a precise gene-editing tool for studying gene functions as well as improving agricultural traits in various crop plants. Here, we optimized a gene-editing system in lettuce (Lactuca sativa L.) using the endogenous U6 promoter and proved that the PHOT2 gene is a versatile target gene. We isolated the LsU6-10 promoter from 10 U6 snRNA genes identified from the lettuce genome database for comparison with the AtU6-26 promoter that has been used to drive sgRNAs in lettuce. Two CRISPR/Cas9 vectors were constructed using the LsU6-10 and AtU6-26 promoters to drive sgRNA361 to target the PHOT2 gene. The chloroplast avoidance response was defective in lettuces with biallelic mutations in the targeted PHOT2 gene, as in the Arabidopsis phot2 mutant. The PHOT2 gene mutations were stably heritable from the R0 to R2 generations, and the high gene-editing efficiency enabled the selection of transgene-free lines in the R1 generation and the establishment of independent phot2 mutants in the R2 generation. Our results suggest that the LsU6-10 promoter is more effective than the AtU6-26 promoter in driving sgRNA for the CRISPR/Cas9 system in lettuce and that PHOT2 is a useful target gene to verify gene editing efficiency without any detrimental effects on plant growth, which is often a consideration in conventional target genes.

Establishment of CRISPR/Cas9 Genome-Editing System Based on Dual sgRNAs in Flammulina filiformis.

Flammulina filiformis, previously known as Asian Flammulina velutipes, is one of the most commercially important edible fungi, with nutritional value and medicinal properties worldwide. However, precision genome editing using CRISPR/Cas9, which is a revolutionary technology and provides a powerful tool for molecular breeding, has not been established in F. filiformis. Here, plasmids harboring expression cassettes of Basidiomycete codon-optimized Cas9 and dual sgRNAs targeting pyrG under the control of the gpd promoter and FfU6 promoter, respectively, were delivered into protoplasts of F. filiformis Dan3 strain through PEG-mediated transformation. The results showed that an efficient native U6 promoter of F. filiformis was identified, and ultimately several pyrG mutants exhibiting 5-fluorooric acid (5-FOA) resistance were obtained. Additionally, diagnostic PCR followed by Sanger sequencing revealed that fragment deletion between the two sgRNA target sites or small insertions and deletions (indels) were introduced in these pyrG mutants through the nonhomologous end joining (NHEJ) pathway, resulting in heritable changes in genomic information. Taken together, this is the first report in which a successful CRISPR/Cas9 genome-editing system based on dual sgRNAs was established in F. filiformis, which broadens the application of this advanced tool in Basidiomycetes.

Expanding the CRISPR Toolbox in Culicine Mosquitoes: In Vitro Validation of Pol III Promoters.

CRISPR-Cas9-based "gene drive" technologies have been proposed as a novel and effective means of controlling human diseases vectored by mosquitoes. However, more complex designs than those demonstrated to date-and an expanded molecular toolbox with which to build them-will be required to overcome the issues of resistance formation/evolution and drive spatial/temporal limitation. Foreseeing this need, we assessed the sgRNA transcriptional activities of 33 phylogenetically diverse insect Polymerase III promoters using three disease-relevant Culicine mosquito cell lines (Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus). We show that U6 promoters work across species with a range of transcriptional activity levels and find 7SK promoters to be especially promising because of their broad phylogenetic activity. We further show that U6 promoters can be substantially truncated without affecting transcriptional levels. These results will be of great utility to researchers involved in developing the next generation of gene drives.

Development of a Bicistronic Vector for the Expression of a CRISPR/Cas9-mCherry System in Fish Cell Lines.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been widely used in animals as an efficient genome editing tool. In fish cells, the technique has been difficult to implement due to the lack of proper vectors that use active promoters to drive the expression of both small guide RNA (sgRNA) and the S. pyogenes Cas9 (spCas9) protein within a single expression platform. Until now, fish cells have been modified using co-transfection of the mRNA of both the sgRNA and the spCas9. In the present study, we describe the optimization of a new vector for the expression of a CRISPR/Cas9 system, designed to edit the genome of fish cell lines, that combines a gene reporter (mCherry), sgRNA, and spCas9 in a single vector, facilitating the study of the efficiency of piscine and non-piscine promoters. A cassette containing the zebrafish U6 RNA III polymerase (U6ZF) promoter was used for the expression of the sgRNA. The new plasmid displayed the expression of spCas9, mCherry, and sgRNA in CHSE/F fish cells. The results demonstrate the functionality of the mammalian promoter and the U6ZF promoter in fish cell lines. This is the first approach aimed at developing a unified genome editing system in fish cells using bicistronic vectors, thus creating a powerful biotechnological platform to study gene function.

Optimization of CRISPR/Cas9 genome editing in cotton by improved sgRNA expression.

BACKGROUND: When developing CRISPR/Cas9 systems for crops, it is crucial to invest time characterizing the genome editing efficiency of the CRISPR/Cas9 cassettes, especially if the transformation system is difficult or time-consuming. Cotton is an important crop for the production of fiber, oil, and biofuel. However, the cotton stable transformation is usually performed using Agrobacterium tumefaciens taking between 8 and 12 months to generate T0 plants. Furthermore, cotton is a heterotetraploid and targeted mutagenesis is considered to be difficult as many genes are duplicated in this complex genome. The application of CRISPR/Cas9 in cotton is severely hampered by the long and technically challenging genetic transformation process, making it imperative to maximize its efficiency. RESULTS: In this study, we provide a new system to evaluate and validate the efficiency of CRISPR/Cas9 cassettes in cotton using a transient expression system. By using this system, we could select the most effective CRISPR/Cas9 cassettes before the stable transformation. We have also optimized the existing cotton CRISPR/Cas9 system to achieve vastly improved mutagenesis efficiency by incorporating an endogenous GhU6 promoter that increases sgRNA expression levels over the Arabidopsis AtU6-29 promoter. The 300 bp GhU6.3 promoter was cloned and validated using the transient expression system. When sgRNAs were expressed under the control of the GhU6.3 promoter in CRISPR/Cas9 cassettes, expression levels were 6-7 times higher than those provided by the AtU6-29 promoter and CRISPR/Cas9-mediated mutation efficiency was improved 4-6 times. CONCLUSIONS: This study provides essential improvements to maximize CRISPR/Cas9-mediated mutation efficiency by reducing risk and workload for the application of CRISPR/Cas9 approaches in the targeted mutagenesis of cotton.

Identification and validation of a Schistosoma japonicum U6 promoter.

BACKGROUND: RNA polymerase III promoters have been widely used to express short hairpin-RNA (shRNA), microRNA (miRNA), and small guide RNA (sgRNA) in gene functional analysis in a variety of organisms including Schistosoma mansoni. However, no endogenous RNA polymerase III promoters have been identified in Schistosoma japonicum. The lack of appropriate promoters in S. japonicum has hindered its gene functional analysis. Identification of functional promoters in S. japonicum is therefore in urgent need. RESULTS: Via sequence alignment, a 347 bp sequence upstream from the coding region of S. japonicum U6 small nuclear RNA (snRNA) was identified, cloned, and named as S. japonicum U6 (sjU6) promoter. A sgRNA sequence named as sgRNA970 was designed, and its Cas9 nuclease guiding activity was confirmed by in vitro cleavage assay. The sjU6 promoter was ligated with sgRNA970 coding sequence by overlap PCR to generate a sjU6-sgRNA970 expression cassette. The expression cassette was inserted into a lentiviral plasmid to construct the pHBLV-sgRNA970 plasmid. First, we tested the sjU6 promoter activity in HEK293 cells by transfecting HEK293 cells with the pHBLV-sgRNA970 plasmid. RT-PCR amplification of the total RNA from the transfected HEK293 cells confirmed the presence of sgRNA970 transcript and indicated sjU6 promoter was functional to initiate transcription in HEK293 cells. Then we transduced the lentivirus expressing Cas9-ZsGreen fusion protein into 14 dpi schistosomula to test whether lentivirus was capable to induce exogenous gene expression in S. japonicum. Fluorescence microscopy and western blot results confirmed the expression of Cas9-ZsGreen fusion protein in S. japonicum. Therefore, this lentiviral system was adapted to test promoter activity in S. japonicum. Finally, we transduced 14 dpi S. japonicum with lentivirus produced from the pHBLV-sgRNA970 plasmid. RT-PCR amplification of the total RNA from transduced schistosomula confirmed the presence of sgRNA970 transcript and therefore indicated sjU6 promoter was functional to initiate transcription in S. japonicum. CONCLUSION: To our knowledge, sjU6 promoter would be the first identified and validated endogenous RNA polymerase III promoter in S. japonicum, which could be used for future CRISPR/Cas9 studies in S. japonicum.

Genome Editing in Diatoms Using CRISPR-Cas to Induce Precise Bi-allelic Deletions.

Genome editing in diatoms has recently been established for the model species Phaeodactylum tricornutum and Thalassiosira pseudonana. The present protocol, although developed for T. pseudonana, can be modified to edit any diatom genome as we utilize the flexible, modular Golden Gate cloning system. The main steps include how to design a construct using Golden Gate cloning for targeting two sites, allowing a precise deletion to be introduced into the target gene. The transformation protocol is explained, as are the methods for screening using band shift assay and/or restriction site loss.