Small Amplicons Mutation Library for Vaccine Screening by Error-Prone Polymerase Chain Reaction

Library construction is a common method used to screen target genes in molecular biology.Most library constructions are not suitable for a small DNA library(<100 base pair(bp))and low RNA library output.To maximize the library’s complexity,error-prone polymerase chain reaction(PCR)was used to increase the base mutation rate.After introducing the DNA fragments into the competent cell,the library complexity could reach 109.Library mutation rate increased exponentially with the dilution and amplification of error-prone PCR.The error-prone PCR conditions were optimized including deoxyribonucleotide triphosphate(dNTP)concentration,Mn^(2+)concentration,Mg^(2+)concentration,PCR cycle number,and primer length.Then,a RNA library with high complexity can be obtained by in vitro transcription to meet most molecular biological screening requirements,and can also be used for mRNA vaccine screening.

Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing

CRISPR/Cas9 uses a guide RNA （gRNA） molecule to execute sequence-specific DNA cleavage and it has been widely used for genome editing in many organisms. Modifications at either end of the gRNAs often render Cas9/gRNA inactive. So far, production of gRNA in vivo has only been achieved by using the U6 and U3 snRNA promoters. However, the U6 and U3 promoters have major limitations such as a lack of cell specificity and unsuitability for in vitro transcription. Here, we present a versatile method for efficiently producing gRNAs both in vitro and in vivo. We design an artificial gene named RGR that, once transcribed, generates an RNA molecule with ribozyme sequences at both ends of the designed gRNA. We show that the primary transcripts of RGR undergo self-catalyzed cleavage to generate the desired gRNA, which can efficiently guide sequence-specific cleavage of DNA targets both in vitro and in yeast. RGR can be transcribed from any promoters and thus allows for cell- and tissue-specific genome editing if appropriate promoters are chosen. Detecting mutations generated by CRISPR is often achieved by enzyme digestions, which are not very compatible with high-throughput analysis. Our system allows for the use of universal primers to produce any gRNAs in vitro, which can then be used with Cas9 protein to detect mutations caused by the gRNAs/CRISPR. In conclusion, we provide a versatile method for generating targeted mutations in specific cells and tissues, and for efficiently detecting the mutations generated.

Epigenetic and Transcriptional Modulator Potential of Epigallocatechin-3-gallate and Genistein on Fetal Hemoglobin Reactivators Genes

Background:𝛽-hemoglobinopathies are one of the most common recessive genetic diseases worldwide,with limited treatments available,particularly in developed countries where the prevalence is higher.Pharmacological reactivation of Fetal Hemoglobin(HbF)is a promising therapeutic strategy.However,approximately 25%of the patients do not respond to Hydroxyurea(HU),the first and most commonly used HbF inducing agent approved by the FDA.Objective:Here,we performed an in vitro assessment of transcriptional effects induced by natural bioactive compounds,namely Epigallocatechin-3-gallate(EGCG)and genistein(GN)in globin genes(HBA1,HBB,HBG1 and HBG2)in HbF regulators/silencer genes(KLF1,BCL11A,MYB and BGLT3)and in epigenetic regulator genes(DNMT1,DNMT3A,DNMT3B,HDAC1,HDAC2,HDAC3 and HDAC8).Moreover,we evaluated EGCG’s in vivo effects in hematological parameters of healthy volunteers.Methods:K562 cells were exposed for 72 and 96 h to GN and EGCG at 100,250 and 500 ng/mL.Cell proliferation and viability were measured,and transcriptional levels were evaluated by qRT-PCR.For in vivo assay,complete blood count was determined by flow cytometry and HbF level was determined through HPLC in 30 healthy individuals before and after 225 mg EGCG ingestion per day during a 90-day period.Results:Both compounds impact cellular metabolism and proliferation with no cytotoxic effects.Divergent GN and EGCG effects in globin and BGLT3 expression levels suggest the involvement of divergent signaling pathways.As for the epigenetic potential,EGCG particularly affects HDAC2 and HDAC8 transcription,whereas GN signifi-cantly affects expression patterns of methylation and acetylation modulators.HU appears to have time divergent effects,with greater impact in methylation at 72 h(overregulates DNTM3A)while affecting acetylation mostly at 96 h(downregulates HDAC1 and HDAC8).Additionally,in vivo,EGCG demonstrated a modulator effect in hematopoiesis and HbF induction.Conclusion:Our results advocate EGCG and GN with HbF pharmacological reactivation potential and sustain further research as new alternative approaches for𝛽-hemoglobinopathies therapies.