Simultaneous precise editing of multiple genes in human cells
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ABSTRACT:
PROVIDER: PRJEB32613 | ENA |
REPOSITORIES: ENA
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Project description:When double-strand breaks are introduced in a genome by CRISPR they are repaired either by non-homologous end joining (NHEJ), which often results in insertions or deletions (indels), or by homology-directed repair (HDR), which allows precise nucleotide substitutions to be introduced if a donor oligonucleotide is provided. Because NHEJ is more efficient than HDR, the frequency with which precise genome editing can be achieved is so low that simultaneous editing of more than one gene has hitherto not been possible. Here, we introduced a mutation in the human PRKDC gene that eliminates the kinase activity of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This results in an increase in HDR irrespective of cell type and CRISPR enzyme used, sometimes allowing 87% of chromosomes in a population of cells to be precisely edited. It also allows for precise editing of up to four genes simultaneously (8 chromosomes) in the same cell. Transient inhibition of DNA-PKcs by the kinase inhibitor M3814 is similarly able to enhance precise genome editing.
| S-EPMC6821318 | biostudies-literature
Project description:RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off- target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking RGNs and high-specificity RGNs both exhibit low off-target activities. Here, we report that high-specificity Cas9 nucleases are convertible into nicking Cas9D10A variants whose precision is superior to that of the commonly used Cas9D10A nickase. Dual nicking RGNs based on a selected group of these Cas9D10A variants can yield gene knockouts and gene knock-ins at frequencies similar to or higher than those achieved by their conventional counterparts. Moreover, high-specificity dual nicking RGNs are capable of distinguishing highly similar sequences by “tiptoeing” over pre-existing single base-pair polymorphisms. Finally, high-specificity RNA-guided nicking complexes generally preserve genomic integrity, as demonstrated by unbiased genome-wide high-throughput sequencing assays. Thus, in addition to substantially enlarging the Cas9 nickase toolkit, we demonstrate the feasibility in expanding the range and precision of genome editing procedures. The herein introduced tools and multi-tier high-specificity genome editing strategies might be particularly beneficial whenever predictability and/or safety of genetic manipulations are paramount.
2020-12-08 | GSE153471 | GEO
Project description:Purpose: A method for mapping chromatin accessibility genome-wide, to reveal chromatin accessibility in Intestinal stem cells. Methods: Intestinal stem cells(Lgr5-high cells) were sorted by flow cytometry from wild type mice. The samples were prepared in duplicate. HISAT2 was used to align the sequences to the mouse genome and generate bam files. bamCoverage was used to generate bigwig files from bam files. MACS2 (v2.2.5) was used for peak calling and to generate bed files from aligned reads. Conclusions: ATAC-seq analysis confirmed that Fosb binding sites in Chip-seq assay were correlated with the chromatin accessibility .
2023-04-13 | GSE186916 | GEO
Project description:Homo sapiens BAM files from amplicon sequencing pipeline
| PRJNA326019 | ENA
Project description:BAM files of waterbuck samples used in PCAngsd
| PRJEB28089 | ENA
Project description:Stroma extracts were isolated from 2-week-old WT plants and incubated with either BSF-specific antibodies or with the pre-immune serum. IgGs were captured with SiMAG-Protein G beads (Chemicell) and recovered RNA was used for generation of libraries with the ScriptSeq v2 RNA-seq Library Preparation Kit (Epicentre). Primary reads were aligned to the Arabidopsis chloroplast genome (accession number NC_000932.1) using CLC Genomics Workbench. BAM files were extracted and sorted in Galaxy . Sorted BAM files were converted into RPKM-normalized bigwig files and displayed in IGB. The differential enrichment of BSF/control of the two replicates was displayed across the entire chloroplast genome to identify the RNA targets of BSF.
2019-04-06 | GSE129411 | GEO