Recent advances in CRISPR-related transposable elements / 生物工程学报

A new wave of research has been inspired by the CRISPR-Cas system with respect to their application in genome editing. The CRISPR-Cas system can not only be applied in gene knockout and insertion, but also be used in base editing, transcriptional regulation and recombination of gene clusters. However, the low efficiency of homology-directed repair (HDR) limits its application. Unlike the CRISPR-Cas system, mobile genetic elements (MGE) can insert DNA fragments into cell chromosomes without the aid of HDR. Recently, it is reported that CRISPR-related transposable elements can guide targeted DNA insertion. Their transposition mechanisms and reprogramming abilities have brought novel opportunities to the development of this field. This review summarized the research progress and application development of natural CRISPR-related transposable elements in recent years, as well as the applications of fused dCas9-transposase. It proposed the application prospects and potential challenges of CRISPR-related transposable elements in the future, which provided a reference for the development direction of gene editing tools.

The improvewment of DNA library construction in non-crosslinked chromatin immunoprecipitation coupled with next-generation sequencing / 南方医科大学学报

OBJECTIVE@#To optimize DNA library construction in non-crosslinked chromatin immunoprecipitation coupled with next-generation sequencing (Native ChIP-seq) to obtain high-quality Native ChIP-seq data.@*METHODS@#Human nasopharyngeal carcinoma HONE1 cell lysate was digested with MNase for release of the nucleosomes, and the histone-DNA complexes were immunoprecipitated with specific antibodies. The protein component in the precipitate was digested with proteinase K followed by DNA purification; the DNA library was constructed for sequence analysis.@*RESULTS@#Compared with the conventional DNA library construction, Tn5 transposase method allowed direct enrichment of the target DNA after Tn5 fragmentation, which was simple, time-saving and more efficient. The IGV visualized map showed that the information obtained by the two library construction methods was consistent. The sequencing data obtained by the two methods revealed more signal enrichment with Tn5 transposase library construction than with the conventional approach. H3K4me3 ChIP results showed a good reproducibility after Tn5 transposase library construction with a signal-to-noise ratio above 50%.@*CONCLUSIONS@#Tn5 transposase method improves the efficiency of DNA library construction and the results of subsequent sequence analysis, and is especially suitable for detecting histone modification in the DNA to provide a better technical option for epigenetic studies.

Using ATAC-seq to identify the chromatin accessibility activated by type I interferon in human monocytes / 上海交通大学学报（医学版）

Objective • To detect the genome-wide profiling of chromatin accessibility in human monocytes after stimulated with interferon α (IFNα). Methods • Blood samples were collected from a healthy donor. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) technique was performed to detect the chromatin accessibility. Bioinformatic tools were used for enrichment analysis and visual analysis. Results • With the treatment of IFNα, there were 430 significant up-regulated regions, and 442 significant down-regulated regions. Most of the accessible regions were located at promoters and the adjacent areas of the genes, followed by the intergenic areas and introns. The enrichment analysis showed that the genes related with up-regulated regions were enriched to interferon relevant pathways or anti-virus reactions. To visualize the corresponding chromatin regions, it showed that the intensity of ATAC-seq signal was significantly enhanced at the promoters and transcriptional start sites of effect or interferon-stimulated genes (ISGs) after IFNα stimulation; while for the regulatory ISGs, there was a certain degree of accessibility before stimulation, and the signal intensity was mildly improved. The motif analysis showed significant enrichment of interferon-stimulated response element and interferon regulatory factor in up-regulated regions. Conclusion • Chromatin accessibility of human monocytes has characteristic changes after typeinterferon stimulation and makes preparation for downstream gene expression.

Establishment of sperm specific Sleeping Beauty transposase-expressing transgenic mouse / 中国比较医学杂志

Objective To establish the sperm specific Sleeping Beauty ( SB ) transposase-expression transgenic mouse for the study of the genetic modification mediated by transposon system in mouse .Methods Prm1 promoter was cloned from mouse genomic DNA to drive the expression of SB transposase .The transgenic mice were generated by microinjection .The gene type of transgenic line was identified by PCR .The expressing level in testis was determined by western blot and immunohistochemistry (IHC) staining.Results Five lines of transposase transgenic mice were obtained by microinjection and three can be germline .One mouse line with higher expression level of transposase in the testis was obtained.Conclusion One transgenic mouse model with Sleeping Beauty transposase - expression was successfully established .This model will greatly contribute to the research of genetic modification mediated by transposon in mouse.

Sequence diversity and copy number variation of Mutator-like transposases in wheat

Partial transposase-coding sequences of Mutator-like elements (MULEs) were isolated from a wild einkorn wheat, Triticum urartu, by degenerate PCR. The isolated sequences were classified into a MuDR or Class I clade and divided into two distinct subclasses (subclass I and subclass II). The average pair-wise identity between members of both subclasses was 58.8 percent at the nucleotide sequence level. Sequence diversity of subclass I was larger than that of subclass II. DNA gel blot analysis showed that subclass I was present as low copy number elements in the genomes of all Triticum and Aegilops accessions surveyed, while subclass II was present as high copy number elements. These two subclasses seemed uncapable of recognizing each other for transposition. The number of copies of subclass II elements was much higher in Aegilops with the S, Sl and D genomes and polyploid Triticum species than in diploid Triticum with the A genome, indicating that active transposition occurred in S, Sl and D genomes before polyploidization. DNA gel blot analysis of six species selected from three subfamilies of Poaceae demonstrated that only the tribe Triticeae possessed both subclasses. These results suggest that the differentiation of these two subclasses occurred before or immediately after the establishment of the tribe Triticeae.