Generation and Application of a Versatile CRISPR Toolkit for Mammalian Cell Engineering

CRISPR/Cas9 has revolutionized the field of genome engineering. Yet, as the CRISPR toolbox has rapidly expanded, there remains a need for a comprehensive library of CRISPR/Cas9 reagents that allow users to perform complex cellular and genetic manipulations without requiring labor-intensive generation of reagents to meet each user’s unique experimental circumstances. Here we described the creation and validation of a pNAX CRISPR library consisting of 72 different Cas9 and gRNA expression plasmids to allow for efficient multiplex gene editing, activation, and repression in mammalian cells. The toolkit plasmids, which are piggyBac or lentiviral based, provide the means for reliable and rapid delivery of Cas9/gRNA through either transient transfection or stable integration. Using the toolkit, we demonstrate the ease with which users can perform single or multiplex gene editing and modulate the expression of both coding and non-coding genes. We also highlight the use of the comprehensive toolkit to perform combinatorial gene knockout to identify factors that regulate homologous recombination, along with investigating the regulatory role of a 68-kb intronic region associated with human disease.

Functional analysis of PCSK9 3′UTR variants and mRNA–miRNA interactions in patients with familial hypercholesterolemia

Aim: Functional analysis of PCSK9 3′UTR variants and mRNA–miRNA interactions were explored in patients with familial hypercholesterolemia (FH). Materials & methods: PCSK9 3′UTR variants were identified by exon-targeted gene sequencing. Functional effects of 3′UTR variants and mRNA–miRNA interactions were analyzed using in silico and in vitro studies in HEK293FT and HepG2 cells. Results: Twelve PCSK9 3′UTR variants were detected in 88 FH patients. c.*75C >T and c.*345C >T disrupted interactions with miR-6875, miR-4721 and miR-564. Transient transfection of the c.*345C >T decreased luciferase activity in HEK293FT cells. miR-4721 and miR-564 mimics reduced PCSK9 expression in HepG2 cells. Conclusion: PCSK9 c.*345C >T has a possible role as loss-of-function variant. miR-4721 and miR-564 downregulate PCSK9 and may be useful to improve lipid profile in FH patients.

miR-34a mimic or pre-mir-34a, which is the better option for cancer therapy? KatoIII as a model to study miRNA action in human gastric cancer cells

Background Aberrantly expressed microRNAs play important roles in gastric tumorigenesis. However, use of miRNAs as a therapeutic option in gastric cancer still remains as a challenging problem. Methods We performed transient transfection of miR-34a-5p mimic and stable transfection of pre-mir-34a into KatoIII cells. Then, we evaluated the effect of transfected miRNAs on numerous cellular and molecular processes. Results Following transient transfection of miR-34a-5p mimic at 25 nM—a commonly used concentration—into KatoIII cells, inhibition of two target genes expression, namely Notch1 and β-catenin, was not observed, but a non-significant marginal increase of these genes was detected. No changes were detected in the percentage of apoptotic cells as well as in CD44 + and EpCAM + cells after 25 nM miR-34a-5p mimic transfection. Interestingly, stable transfection of pre-mir-34a into KatoIII cells (named as KatoIII-pGFPC1-34a cells) caused a significant repression in β-catenin protein and Notch1 mRNA levels (p < 0.05 and p < 0.01, respectively) relative to equivalent control (KatoIII- pGFPC1-empty cells). The percentage of CD44 + cells in the KatoIII-pGFPC1-34a cells (< 40%) was significantly lower than that in control cells (~ 95%) (p < 0.05). An increase of ~ 3.5% in apoptotic cells and a slower proliferation rate were detected in KatoIII-pGFPC1-34a cells. Conclusions Our study revealed that the effect of miR mimic in target gene repression can be dependent to its concentration as well as to the cell type. Meanwhile, our findings further support a regulatory function for pre-miRNAs in target repression and will help to develop effective therapeutic strategies in cancer treatment.

Natural Variation of Dt2 Promoter Controls Plant Height and Node Number in Semi-Determinant Soybeans

Soybean [Glycine max (L.) Merrill] is one of the most important legume crops and its plant height (PH) is one significant quantitative trait closely related to node number (NN) and internode length (IL) on the main stem, which affect yield together. In this study, we used a recombinant inbred line (RIL) derived from a cross between two semi-determinate stem soybean varieties (Dt1Dt1Dt2Dt2), JKK378 and HXW. A consistent QTL named qPH18 simultaneously controlling PH, NN, and IL was identified, in which this region harbors the semi-determinant gene, Dt2. Sequencing of the promoter of Dt2 from JKK378, identified three polymorphisms relative to HXW, including two SNPs and a 18bp Indel. The expression level of Dt2 in qPH18JKK378 group was lower than that in qPH18HXW group, meanwhile, as a downstream gene, the expression of Dt1 from two groups showed a contrary tendency. Transient transfection assay confirmed that the promoter activity of Dt2 from JKK378 is lower compared to HXW. We speculate that the polymorphisms in the two dominant Dt2 promoter caused difference in expression level of Dt2 and its downstream gene Dt1, hence, affect PH, NN, IL, and grain weight per plant without changing stem growth habit. Compared to PH18HXW allele, qPH18JKK378 allele suppresses the expression of Dt2 gene which releases the expression of Dt1, thus promotes plant node number and enhanced grain yield. These results will be helpful for revealing the mechanism underlying the node number and plant height, the soybean materials and molecular marker will facilitate molecule breeding.

Rabies virus glycoprotein produced in Nicotiana benthamiana is an immunogenic antigen in mice

Rabies remains an infectious disease among humans and animals, and requires the development of an effective vaccine essential to prevent rabies. Advances in molecular biology and biotechnology have led to the development and improvement of many rabies vaccines. Before the third-generation of the vaccine, rabies vaccines were based on the virus itself. Thus, even if effective, these vaccines may not be completely safe, resulting in a strong demand for the development of effective subunit vaccines that do not raise concerns about virus replication and infection in the host. This study investigated the ability of the glycoprotein of the rabies virus to be expressed in tobacco plants (Nicotiana benthamiana) and to induce an immune response in mice. Using a transient transfection, a soluble glycoprotein was successfully expressed in N. benthamiana. Fusing of five histidine residues at the C-terminus enabled the glycoprotein to be easily purified by affinity chromatography. The glycoprotein expressed in the plants was found to be N-glycosylated post-translationally, and the mice immunised with this glycoprotein generated neutralising antibodies against the rabies virus. These results suggest that a glycoprotein produced in the endoplasmic reticulum of N. benthamiana is bioactive, and might be used to generate a subunit vaccine against the rabies virus.