SNPs in miRNAs and Target Sequences: Role in Cancer and Diabetes

miRNAs are fascinating molecular players for gene regulation as individual miRNA can control multiple targets and a single target can be regulated by multiple miRNAs. Loss of miRNA regulated gene expression is often reported to be implicated in various human diseases like diabetes and cancer. Recently, geneticists across the world started reporting single nucleotide polymorphism (SNPs) in seed sequences of miRNAs. Similarly, SNPs are also reported in various target sequences of these miRNAs. Both the scenarios lead to dysregulated gene expression which may result in the progression of diseases. In the present paper, we explore SNPs in various miRNAs and their target sequences reported in various human cancers as well as diabetes. Similarly, we also present evidence of these mutations in various other human diseases.

Monitoring of plant raw materials and feed for animal in 2020 for the presence of GM-ingredients

The creation and the use of genetically modified products has become a tendency in the development of agricultural and food technologies. The area of agricultural land under genetically modified plants is constantly growing. Todays the process of using GMOs and the expediency of their creation is a debatable issue. The modification of the genome of traditional agricultural cultures gives them resistance to pesticides, pests, diseases, which cause to the significant an increase of harvest and improved quality and taste characteristics. However, the effects of GMOs on the environment and the body of animals and humans have not been fully studied, and therefore the thoughts of scientists are differ on the benefits and risks of genetic engineering. Recently, the scientific literature has data on the negative effects of GMOs on animals and humans, in particular, on the morphofunctional state of organs and systems of the body, reproductive function, immune status, biochemical parameters of blood and urine. Every year the number of new genetically modified plant lines is growing, so today, the need in research of plant raw materials and feed for animal on the presence of GMOs is very important and actual. The article presents the results of research on the detection of GM ingredients in plant raw materials and in products of its processing, feed for productive and unproductive animals, etc. In 2020, 1215 samples were investigated by polymerase chain reaction with detection in real-time (PCR-RT), and it was found out that only 0.3 % from total amount were positive. From the studied samples, the most positive samples were found in samples of rapeseed, soybeans and feed for productive animals. In 27 samples of rapeseed, the number of positive samples was 7.4 %, in them were detected the target sequences of the terminator NOS (T-NOS) T plasmid Agrobacterium, and genes Pat and EPSPs. In 6 samples of soybean, the number of positive samples was 16.7 %, in them were detected the target sequences of the 35S promoter of cauliflower mosaic virus (CaMV) and the terminator NOS (T-NOS) T of the plasmid Agrobacterium. Also there was found GM ingredients in compound feeds for farm animals and poultry, in 6 samples the number of positive samples was 16.7 %, in them were detected the target sequences of the terminator NOS (T-NOS) T plasmid Agrobacterium. Conducted studies indicate that transgenic plants are in circulation in the agricultural market, so it is necessary to constantly control animal feed, plant materials and seeds for the presence of GM sources.

EXTH-65. INSERTION OF MICRORNA TARGET SEQUENCES INTO RETROVIRAL REPLICATING VECTORS EFFECTIVELY RESTRICTS TRANSGENE EXPRESSION AND VIRAL REPLICATION IN HUMAN HEMATOPOIETIC STEM AND PROGENITOR CELLS

Prodrug activator gene therapy with a retroviral replicating vector (RRV) has shown a highly favorable safety profile and long-term survival in early-phase trials for recurrent high-grade glioma. Overall endpoints were not met in a recent Phase 3 trial, but highly statistically significant survival was observed in prespecified patient subgroups as compared to randomized matched control patients receiving standard-of-care treatments, and further clinical evaluation is being focused on these subgroups. Additional strategies to enhance therapeutic potency may require two or more RRVs to deliver multiple transgenes simultaneously, but RRVs encoated with the same envelope protein will compete for the same cellular receptors, interfering with efficient co-infection. The current clinical vector (formerly Toca511, now DB107) is encoated by amphotropic murine leukemia virus (MLV) envelope, which binds to inorganic phosphate transporter PiT-2/SLC20A2. To switch RRV tropism, we developed RRVs pseudotyped with a heterologous envelope from Gibbon ape leukemia virus (GALV), which utilizes an alternative phosphate transporter, PiT-1/SLC20A1, for cell entry. Efficient co-infection of established and primary human glioblastoma cells with MLV- and GALV-pseudotyped RRV was achieved, without receptor competition. However, human hematopoietic stem/progenitor cells (HSPC) also express high levels of PiT-1, which may increase potential genotoxicity of GALV-pseudotyped RRV. Accordingly, as a novel strategy to restrict gene expression and replication in HSPC, we have developed and tested new RRV designs incorporating microRNA target sequences (miRT). Insertion of miRT recognized by hematopoietic lineage-specific microRNA-142-3p resulted in complete suppression of RRV replication in primary human CD34+ HSPC, with the percentage of infected cells below 1% after vector inoculation at multiplicities of infection (MOI) 0.05 and 0.1, and remaining below 3% even after inoculation at MOI 0.5 and 1.0. In vivo models have been developed for on-going studies to evaluate miRT-mediated restriction strategies for avoidance of potential genotoxicity by RRV in normal hematopoietic cells.

CRISPR/Cas9 inhibits rather than induces non-targeted DNA cleavage more likely to cause off-target single-nucleotide variants

CRISPR/Cas9 gene targeting technology has become the most widely used gene editing technology in both plants and animals. However, substantial off-target effect remains as a major imperfection hindering its further application. Here, Nicotiana benthamiana leaf cell-free system was used to simulate in vivo environment. And the effects of different CRISPR/Cas9 components on DNA stability in cell-free system were studied to explore possible mechanisms causing CRISPR off-target. The results showed that overexpressing Cas9, nCas9 and dCas9 significantly inhibited DNA cleavage in the cell extracts. While overexpressing RNPs accelerated the target DNA cleavage but inhibited non-target DNA digestion in cell extracts, overexpressing nRNP and dRNP blocked the cleavage of either target or non-target sequences. Meanwhile, analysis of whole-genome sequencing data from mice and rice edited by different CRISPR tools revealed that the main off-target mutations were SNVs (single nucleotide variants), rather than Indels (insertions and deletions) that were readily induced by DNA double-strand breaks. The off-target sites did not match the conventionally predicted places but were PAM-rich sites preferred. Our study suggests that PAM-dependent binding without cleavage of CRISPR/Cas9 to non-target sequences may increase off-target mutation risks through impeding the necessary cleavage process for repairing spontaneous or environmentally induced non-targeted DNA mutations.

Generation of a more efficient prime editor 2 by addition of the Rad51 DNA-binding domain

Although prime editing is a promising genome editing method, the efficiency of prime editor 2 (PE2) is often insufficient. Here we generate a more efficient variant of PE2, named hyPE2, by adding the Rad51 DNA-binding domain. When tested at endogenous sites, hyPE2 shows a median of 1.5- or 1.4- fold (range, 0.99- to 2.6-fold) higher efficiencies than PE2; furthermore, at sites where PE2-induced prime editing is very inefficient (efficiency < 1%), hyPE2 enables prime editing with efficiencies ranging from 1.1% to 2.9% at up to 34% of target sequences, potentially facilitating prime editing applications.

The Potential of U6 and Its Copies in the Regulation of the Human Genome

Non-coding RNAs are conformed by a large repertoire of RNA molecules with unimaginable tridimensional structures and functions. Small nuclear RNAs are an essential part of the spliceosome machinery, which is crucial for proper mRNA maturation. It is important to add that U6, one of the four snRNAs forming the spliceosome has been extensively studied. Full-length U6 (U6-1) loci are widely dispersed throughout the genome (200-900 copies), but a few U6 full-length loci have been identified to date as potentially active genes. The importance of U6 to carry out, together with other snRNAs, the catalytic activity and recognition of annealing target sequences, its evolution in the genome and the fact that the genome has many U6 copies and pseudogenes, its association with retrotransposition, as well as their implication in diseases is discussed in this review.

Cytomegalovirus late transcription factor target sequence diversity orchestrates viral early to late transcription

Beta- and gammaherpesviruses late transcription factors (LTFs) target viral promoters containing a TATT sequence to drive transcription after viral DNA replication has begun. Human cytomegalovirus (HCMV), a betaherpesvirus, uses the UL87 LTF to bind both TATT and host RNA polymerase II (Pol II), whereas the UL79 LTF has been suggested to drive productive elongation. Here we apply integrated functional genomics (dTag system, PRO-Seq, ChIP-Seq, and promoter function assays) to uncover the contribution of diversity in LTF target sequences in determining degree and scope to which LTFs drive viral transcription. We characterize the DNA sequence patterns in LTF-responsive and -unresponsive promoter populations, determine where and when Pol II initiates transcription, identify sites of LTF binding genome-wide, and quantify change in nascent transcripts from individual promoters in relation to core promoter sequences, LTF loss, stage of infection, and viral DNA replication. We find that HCMV UL79 and UL87 LTFs function concordantly to initiate transcription from over half of all active viral promoters in late infection, while not appreciably affecting host transcription. Both LTFs act on and bind to viral early-late and late kinetic-class promoters. Over one-third of these core promoters lack the TATT and instead have a TATAT, TGTT, or YRYT. The TATT and non-TATT motifs are part of a sequence block with a sequence code that correlates with promoter transcription level. LTF occupancy of a TATATA palindrome shared by back-to-back promoters is linked to bidirectional transcription. We conclude that diversity in LTF target sequences shapes the LTF-transformative program that drives the viral early-to-late transcription switch.

SAUTE: sequence assembly using target enrichment

Background Illumina is the dominant sequencing technology at this time. Short length, short insert size, some systematic biases, and low-level carryover contamination in Illumina reads continue to make assembly of repeated regions a challenging problem. Some applications also require finding multiple well supported variants for assembled regions. Results To facilitate assembly of repeat regions and to report multiple well supported variants when a user can provide target sequences to assist the assembly, we propose SAUTE and SAUTE_PROT assemblers. Both assemblers use de Bruijn graph on reads. Targets can be transcripts or proteins for RNA-seq reads and transcripts, proteins, or genomic regions for genomic reads. Target sequences are nucleotide and protein sequences for SAUTE and SAUTE_PROT, respectively. Conclusions For RNA-seq, comparisons with Trinity, rnaSPAdes, SPAligner, and SPAdes assembly of reads aligned to target proteins by DIAMOND show that SAUTE_PROT finds more coding sequences that translate to benchmark proteins. Using AMRFinderPlus calls, we find SAUTE has higher sensitivity and precision than SPAdes, plasmidSPAdes, SPAligner, and SPAdes assembly of reads aligned to target regions by HISAT2. It also has better sensitivity than SKESA but worse precision.