ABSTRACT
The viral genome titer is a crucial indicator for the clinical dosing, manufacturing, and analytical testing of recombinant adeno-associated virus (rAAV) gene therapy products. Although quantitative PCR and digital PCR are the common methods used for quantifying the rAAV genome titer, they are limited by inadequate accuracy and robustness. The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a biosensor is being increasingly used in virus detection; however, there is currently no report on its application in the titer determination of gene therapy products. In the present study, an amplification-free CRISPR-Cas12a assay was developed, optimized, and applied for rAAV genome titer determination. The assay demonstrated high precision and accuracy within the detection range of 4 × 109 and 1011 vg/mL. No significant difference was observed between the Cas12a and qPCR assay results (p < 0.05, t test). Moreover, Cas12a exhibited similar activity on both single-stranded and double-stranded DNA substrates. Based on this characteristic, the titers of positive-sense and negative-sense strands were determined separately, which revealed a significant difference between their titers for an in-house reference AAV5-IN. This study presents the inaugural report of a Cas12a assay developed for the titer determination and composition analysis of the rAAV genome.
ABSTRACT
BACKGROUND: Tartary buckwheat (Fagopyrum tataricum), an important pseudocereal crop, has high economic value due to its nutritional and medicinal properties. However, dehulling of Tartary buckwheat is difficult owing to its thick and tough hull, which has greatly limited the development of the Tartary buckwheat processing industry. The construction of high-resolution genetic maps serves as a basis for identifying quantitative trait loci (QTLs) and qualitative trait genes for agronomic traits. In this study, a recombinant inbred lines (XJ-RILs) population derived from a cross between the easily dehulled Rice-Tartary type and Tartary buckwheat type was genotyped using restriction site-associated DNA (RAD) sequencing to construct a high-density SNP genetic map. Furthermore, QTLs for 1000-grain weight (TGW) and genes controlling hull type were mapped in multiple environments. RESULTS: In total, 4151 bin markers comprising 122,185 SNPs were used to construct the genetic linkage map. The map consisted of 8 linkage groups and covered 1444.15 cM, with an average distance of 0.35 cM between adjacent bin markers. Nine QTLs for TGW were detected and distributed on four loci on chromosome 1 and 4. A major locus detected in all three trials was mapped in 38.2-39.8 cM region on chromosome 1, with an LOD score of 18.1-37.0, and explained for 23.6-47.5% of the phenotypic variation. The genes controlling hull type were mapped to chromosome 1 between marker Block330 and Block331, which was closely followed by the major locus for TGW. The expression levels of the seven candidate genes controlling hull type present in the region between Block330 and Block336 was low during grain development, and no significant difference was observed between the parental lines. Six non-synonymous coding SNPs were found between the two parents in the region. CONCLUSIONS: We constructed a high-density SNP genetic map for the first time in Tartary buckwheat. The mapped major loci controlling TGW and hull type will be valuable for gene cloning and revealing the mechanism underlying grain development and easy dehulling, and marker-assisted selection in Tartary buckwheat.
Subject(s)
Fagopyrum , Edible Grain , Fagopyrum/genetics , Genetic Linkage , Polymorphism, Single Nucleotide , Quantitative Trait LociABSTRACT
FSH plays a key role in the function of the reproductive system of human beings and is widely used both diagnostically and therapeutically in reproductive medicine. With the growing incidence of infertility, the demand for FSH pharmaceutical products is increasing. For this reason, the quality control process for FSH products is becoming more stringent. An accurate determination of bioactivity is crucial for the safety and efficacy of recombinant human follicle stimulating hormone (rhFSH). Up to now, in-vivo bioassay based on FSH-induced increases in rat ovarian weight has been the only method widely accepted by different pharmacopoeias. However this method has such drawbacks as the complex procedures, long assay period and high variability. Here, we established a reporter gene assay (RGA) based on the CHO-K1-FSHR-CRE-Luc cell line that stably expresses human follicle stimulating hormone receptor (hFSHR), as well as a luciferase reporter under the control of cyclic adenosine monophosphate (cAMP) response elements (CRES). Our study showed that our new assay not only has good dose-dependent responsiveness to rhFSH, but it also performs excellently in terms of specificity, precision, linearity, and simplicity compared with in-vivo rat bioassays. These results implied that this robust reporter gene assay may be a viable supplement to the animal in-vivo bioassay and may be employed in potency determination of rhFSH pharmaceutical products.