ABSTRACT: CRISPR/Cas9-mediated enrichment coupled to nanopore sequencing provides a valuable tool for the precise reconstruction of large genomic target regions
Project description:By a robust unbiased ChIP-seq approach, we demonstrated that CRISPR/Cas9 had crRNA-specific off-target binding activities in human genome. However, most of those binding off-targets could not be efficiently cleaved both in vivo and in vitro which suggested the cleavage off-target activity of CRISPR/Cas9 in human genome is very limited. We provided a valuable tool to further investigate the molecular mechanism of CRISPR/Cas9 and to optimize its in vivo targeting sgRNA binding sites were identified with ChipSeq by using GFP antibody (there are 2 replicates for egfa-t1 sgRNA,emx1 sgRNA and control without sgRNA in Hek293T cells, one egfa-t1 sgRNA,emx1 sgRNA and control without sgRNA in HeLaS3 cells)
Project description:Metastatic prostate cancer remains a major clinical challenge. Liquid biopsy is emerging as a promising tool in disease management and provides valuable opportunities to gain insights into the underlying biology. Here, we used the highly sensitive, methylated DNA immunoprecipitation coupled to next generation sequencing (MeDIP) technology, to interrogate the cell-free DNA methylomes across localized and metastatic prostate cancer.
| EGAS00001005522 | EGA
Project description:Cas9 targeted enrichment of mobile elements using nanopore sequencing
Project description:We used the nanopore Cas9 targeted sequencing (nCATS) strategy to specifically sequence 125 L1HS-containing loci in parallel and measure their DNA methylation levels using nanopore long-read sequencing. Each targeted locus is sequenced at high coverage (~45X) with unambiguously mapped reads spanning the entire L1 element, as well as its flanking sequences over several kilobases. The genome-wide profile of L1 methylation was also assessed by bs-ATLAS-seq in the same cell lines (E-MTAB-10895).
Project description:Non-cleaving Cas9 (dCas9) is widely employed to manipulate specific gene loci often with scant regard for unintended transcriptional effects. We demonstrate here that dCas9 mediates precise transcriptional pausing followed by transcription termination and potential alternative polyadenylation. In contrast, alternative splicing is unaffected, likely requiring more sustained alteration to RNA polymerase II elongation speed. The effect on transcription is orientation-specific, with pausing only being induced when dCas9-associated guide RNA anneals to the non-template strand. Targeting template strand induces minimal effects on transcription elongation and thus provides a neutral approach to recruit dCas9 linked effector domains to specific gene regions. In essence we evaluate molecular effects of targeting dCas9 to mammalian transcription units. In so doing we also provide new information on transcriptional elongation by RNA polymerase II and coupled pre-mRNA processing.
Project description:This dataset contains Xdrop followed by oxford nanopore long read sequencing performed in target tRNA gene deletion clones in HAP1 (t72) and HepG2 (t15). By applying de novo assembly based approach to Xdrop-LRS data, we identified Cas9-induced on-target genomic alteration.
Project description:This dataset contains Xdrop followed by oxford nanopore long read sequencing performed in target tRNA gene deletion (t8) and intergenic region deletion (i50) clones in HepG2 . By applying de novo assembly based approach to Xdrop-LRS data, we identified Cas9-induced on-target genomic alteration.
Project description:Our multi-omics analysis of local patients demonstrated significant distinctions compared to TCGA-THCA cohorts, highlighting the unique genetic characteristics of Chinese individuals with PTC. We identified novel biomarkers that hold potential for diagnosis and prognosis of PTC. Furthermore, developing a logistic model based on the data from local patients provides a valuable tool for precise diagnosis specifically tailored to the Chinese population.
Project description:By a robust unbiased ChIP-seq approach, we demonstrated that CRISPR/Cas9 had crRNA-specific off-target binding activities in human genome. However, most of those binding off-targets could not be efficiently cleaved both in vivo and in vitro which suggested the cleavage off-target activity of CRISPR/Cas9 in human genome is very limited. We provided a valuable tool to further investigate the molecular mechanism of CRISPR/Cas9 and to optimize its in vivo targeting