Benchmarking of SpCas9 variants enables deeper base editor screens of BRCA1 and BCL2
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ABSTRACT:
PROVIDER: PRJNA747943 | ENA |
REPOSITORIES: ENA
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Project description:Numerous rationally-designed and directed-evolution variants of SpCas9 have been reported to expand the utility of CRISPR technology. Here, we benchmark PAM preferences, on-target activity, and off-target susceptibility of 11 variants of SpCas9 in cell culture assays with thousands of guides targeting endogenous genes. To enhance the coverage and thus utility of base editing screens, we demonstrate that the SpCas9-NG and SpG variants are compatible with both A>G and C>T base editors, more than tripling the number of guides and assayable residues. We demonstrate the performance of these technologies by screening for loss-of-function mutations in BRCA1 and Venetoclax-resistant mutations of BCL2, identifying both known and new insights into these clinically-relevant genes. We anticipate that the tools and methodologies described here will facilitate the investigation of genetic variants at a finer and deeper resolution for any locus of interest.
2021-07-21 | GSE180351 | GEO
Project description:Benchmarking of SpCas9 variants enables deeper base editor screens of BRCA1 and BCL2
| PRJNA753064 | ENA
Project description:We identified robust cleavage by non-canonically-targeted SpCas9 that target protospacer with a few bases from the canonical NGG PAM in biochemistry assay. To determine whether this spaced SpCas9 cleavage exists in human cells, we employed high-throughput genome-wide translocation sequencing (LAM-HTGTS) using SpCas9 with three independent canonical sgRNAs served as bait (bait-A, bait-L and bait-D) to compared the cleavage activities of canonically-targeted and non-canonically-targeted SpCas9 in eight locus (RAG1B-D, RAG1K-O) in chromosome 11. We found that almost all 1bp-target shifted SpCas9 generated substantial DSBs as the canonical controls. While detected less frequently, 2bp-targeted shifted SpCas9 generated significant amounts of DSBs in RAG1D and RAG1L locus. These findings underscore the spaced PAM-mediated DSBs occurred in living cells.
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Project description:In this study, we used a surrogate lentivirus library to capture CRISPR editing outcome in HEK cells. The dataset include quantification of indel frequencies for SpCas9 gRNAs in 12,000 surrogate sites. After filtering low quality sites, the high quality SpCas9 gRNA activities from a total of 10592 sites have been used to develop an improved deep learning-based prediction model CRISPRon (https://rth.dk/resources/crispr/.).
2021-05-03 | GSE173708 | GEO
Project description:We describe an improved individual nucleotide resolution CLIP protocol (iiCLIP), which can be completed within 4 days from UV crosslinking to libraries for sequencing. For benchmarking, we directly compared PTBP1 iiCLIP libraries with the iCLIP2 protocol produced under standardised conditions with 1 million HEK293 cells, and with public eCLIP and iCLIP PTBP1 data. There are 3 PTBP1 iiCLIP libraries, 1 input iiCLIP library and 1 PTBP1 iCLIP2 library produced in this study.
2022-08-01 | E-MTAB-10881 | biostudies-arrayexpress
Project description:Optimization of CRISPR/Cas9-mediated genome engineering has resulted in base editors that hold promise for mutation repair and disease modeling. Here, we demonstrate the application of base editors for the generation of complex tumor models in human ASC-derived organoids. First we show Efficacy of cytosine and adenine base editors in modelingCTNNB1hot-spot mutations in hepatocyte organoids. Next, we use C>T base editors to insert nonsense mutations inPTENin endometrial organoids and demonstrate tumorigenicity even in the heterozygous state. Moreover, drug screening assays on organoids harboring eitherPTENorPTENandPIK3CAmutations reveal the mechanism underlying the initial stages of endometrial tumorigenesis. To further increase the scope of base editing we combine SpCas9 and SaCas9 for simultaneous C>T and A>G editing at individual target sites. Finally, we show that base editor multiplexing allow modeling of colorectal tumorigenesis in a single step by simultaneously transfecting sgRNAs targeting five cancer genes.
2023-07-30 | GSE236490 | GEO
Project description:Brain organoids (BO) enabled the investigation of human corticogenesis in-vitro with an increasing range of protocols achieving its remarkable recapitulation. However, we lack a resource gathering fetal cortex-specific gene co-expression patterns and their behavior in BO. We complement the current knowledge with a benchmarking of BO versus human corticogenesis, integrating: transcriptomes from in-house differentiated cortical BO (CBO), in-house processed human fetal brain samples, analysis of transcriptomes from different BO systems and of pre-natal cortical samples from the BrainSpan Atlas.
2022-12-31 | E-MTAB-11239 | biostudies-arrayexpress
Project description:Activation of the MYC oncogene is common in B-cell lymphomas, and frequently associated with compensatory events that dampen Myc-induced apoptosis, such as over-expression of anti-apoptotic Bcl2-family proteins. For example, concurrent translocations of MYC and BCL2 in a subset of Diffuse large B-cell lymphoma (DLBCL) lead to the high-grade “double-hit” lymphoma subtype (DHL), characterized by dismal prognosis in the face of current front-line regimens, thus calling for the pursuit of tailored therapeutic strategies. Here, we show that Myc and Bcl2 modulate the sensitivity of B-cells to IACS-010759, a selective inhibitor of mitochondrial respiratory complex I. Myc activation in non-transformed lymphoid precursors suppressed endogenous Bcl2 and sensitized the cells to IACS-010759-induced apoptosis. Treatment with the Bcl2 inhibitor venetoclax also sensitized to IACS-010759, while overexpression of Bcl-2 was protective. IACS-010759 engaged an ATF4-driven Integrated Stress Response (ISR) with dual anti- and pro-apoptotic effects, the latter mediated by the CHOP transcription factor, which contributed to selective killing of Myc-overexpressing cells. In line with the above data, IACS-010759 and venetoclax synergized in killing human DHL cells, and showed strong combinatorial effects in a pre-clinical setting. In a Bcl2-negative Burkitt’s lymphoma cell line, instead, IACS-010759 synergized with the Mcl-1 inhibitor S63845. Altogether, our data point to the combination of IACS-010759 with distinct Bcl2-family inhibitors for therapeutic reactivation of the intrinsic apoptotic pathway in Myc-associated B-cell lymphomas
2022-03-01 | GSE149073 | GEO