Precise and broad scope genome editing based on high-specificity Cas9 nickases
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ABSTRACT:
PROVIDER: PRJNA642789 | ENA |
REPOSITORIES: ENA
Similar Datasets
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: Not available
| S-EPMC7826261 | biostudies-literature
Project description:Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor, and surgical resection is a key part of the standard-of-care. In fluorescence-guided surgery (FGS), fluorophores are used to differentiate tumor tissue from surrounding normal brain. The heme synthesis pathway converts 5-aminolevulinic acid (5-ALA), a fluorogenic substrate, to the fluorophore protoporphyrin IX (PpIX). The resulting fluorescence is thought to be specific to transformed glioma cells, but this specificity has not been examined at single cell level. Here, we performed paired single cell imaging and RNA sequencing of individual cells (SCOPE-seq2) on human GBM surgical specimens with visible PpIX fluorescence from patients who received 5-ALA prior to surgery. SCOPE-seq2 allows us to simultaneously measure PpIX fluorescence by imaging and unambiguously identify transformed glioma cells from single-cell RNA-seq (scRNA-seq). We observed that 5-ALA treatment results in labeling that is not specific to transformed tumor cells. In cell culture experiments, we further demonstrated untransformed cells can be labeled by 5-ALA directly or by PpIX secreted from surrounding transformed cells. In acute slice cultures from mouse glioma models, we showed that 5-ALA preferably labels GBM tumor tissue over non-neoplastic brain tissue at bulk level, and that this contrast is not due to blood-brain-barrier disruption. Taken together, our findings support the use of 5-ALA as an indicator of GBM tissue, but not as a specific marker of transformed glioma cells.
2022-11-30 | GSE218331 | GEO
Project description:Despite huge efforts to map the human proteome using mass spectrometry the overall sequence coverage achieved to date is still below 50%. Reasons for missing areas of the proteome comprise protease-resistant domains including the lack/excess of enzymatic cleavage sites, non-unique peptide sequences, impaired peptide ionization/separation and low expression levels. To access novel areas of the proteome the beneficial use of enzymes complementary to trypsin, such as GluC, AspN, LysN, ArgC, LysArginase has been reported. Here, we present how the broad-specificity protease subtilisin enables mapping of previously hidden areas of the proteome. We systematically evaluated its digestion efficiency and reproducibility and compared it to the gold standard in the field, trypsin. Notably, subtilisin allows reproducible near-complete digestion of cells lysates in 1 minute. As expected from its broad specificity the generation of overlapping peptide sequences reduces the number of identified proteins compared to trypsin (8,363 vs. 6,807; 1% protein FDR). However, subtilisin considerably improved the coverage of missing and particularly proline-rich areas of the proteome. Along 14,628 high confidence phosphorylation sites identified in total, only 33% were shared between both enzymes, while 37% were exclusive to subtilisin. Notably, 926 of these were not even accessible by additional in silico digestion with either AspN, ArgC, GluC, LysC, or LysN. Thus, subtilisin might be particularly beneficial for system-wide profiling of post-translational modification sites. Finally, we demonstrate that subtilisin can be used for reporter-ion based in-depth quantification, providing a precision comparable to trypsin – despite broad specificity and fast digestion that may increase technical variance.
2017-11-30 | PXD008068 | Pride
Project description:Skin samples were analyzed through Seq-Scope.
2022-07-22 | GSE186601 | GEO
Project description:We have devised fast and robust SP3- and STRAP-based protocols for the broad-specificity proteases subtilisin, proteinase K, and thermolysin. All three enzymes are remarkably fast, producing near-complete digests in 1-5 min, and cost 200-1000x less than proteomics-grade trypsin. Using FragPipe resolved a major challenge by drastically reducing the duration of the required "unspecific" searches.
In-depth analyses of proteinase K, subtilisin, thermolysin Jurkat digests identified 7374, 8178, 8752 unique proteins with average sequence coverages of 21%, 29%, 37%, including tens of thousands of amino acids not reported in the PeptideAtlas database spanning over 2400 experiments. While we could not identify distinct cleavage patterns, machine learning could distinguish true protease products from random cleavages, potentially enabling the prediction of cleavage products.
2D runs: 20 high-pH RP-HPLC fractions analyzed by DDA LC-MS/MS mode on a timsTOF Pro2, with Evosep; 15 SPD method, 200 ng peptide loaded on-column.
STRAP vs SP3: each digest prepared in triplicate with SP3 and STRAP, respectiveyl-- analyzed by DDA nano-LC-MS/MS on an Orbitrap Exploris 480, 87 min gradient, 750 ng peptide loaded on-column.
2023-11-29 | MSV000093532 | MassIVE
Project description:The hpCasMINI exhibits high specificity for gene editing. Compared to SpCas9 and LbCas12a, hpCasMINI also exhibited higher specificity for genomic DNA cleavage at the tested sites.
2024-06-29 | GSE270588 | GEO
Project description: Not available
| S-EPMC7461344 | biostudies-literature
Project description: Not available
| S-EPMC6548941 | biostudies-literature
Project description:In eukaryotic cells, protein synthesis typically begins with the binding of eIF4F to the 7-methylguanylate (m7G)cap found on the 5’ end of the majority of mRNAs. Surprisingly, overall translational output remains robust under eIF4F inhibition. The sustained protein synthesis has been largely attributed to cap-independent translation mediated by internal ribosome entry sites (IRES). However, the IRES-driven translation is substrate-specific and largely incompatible with the broad spectrum of eIF4F-resistant translatomes.Here, we report that N6-methyladenosine (m6A)-mediated translation prevails on capped mRNAs and is resistant to eIF4F inactivation.Depletion of the methyltransferase METTL3 selectively inhibits translation of mRNAs bearing 5’UTR methylation, but not mRNAs with 5’ terminal oligopyrimidine (TOP) elements. Mechanistically, we identify ABCF1 as a critical mediator of m6A-promoted translation under both stress and physiological conditions. Supporting the role of ABCF1 in m6A-mediated cap-independent translation, ABCF1-sensitive transcripts largely overlap with METTL3-responsible mRNA targets. By illustrating the scope and the mechanism of translation initiation that is neither cap- nor IRES-dependent, these findingsreshape our current perceptions of cellular translational pathways
2017-07-26 | GSE101865 | GEO