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CHANGE-seq-BE enables simultaneously sensitive and unbiased in vitro profiling of Cas-dependent base editor genome-wide off-target activity IV

ABSTRACT: To assess genome-wide off-target activity of base editors and Cas9 nucleases identified by CHANGE-seq-BE, Digenome-seq, and CHANGE-seq, we performed hybrid capture sequencing for five therupatic loci (B2M, CBLB, CD7, CIITA, PDCD1) in human primary T-cells and PCSK9 in human hepatocytes. We observed high on-target editing for ABE, CBE, Cas9 mRNA edited cells and potential off-targets confirmed by hybrid capture sequencing. Our results demonstrate that CHANGE-seq-BE is highly sensitive than Digenome-seq to detect more bona fide off-targets with cellular activity ranging from 0.5% to 92.2%. Moreover, ABE exhibit more off-targets than Cas9 under same delivery conditions.

ORGANISM(S): Homo sapiens

PROVIDER: GSE307892 | GEO | 2025/11/05

REPOSITORIES: GEO

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CHANGE-seq-BE enables simultaneously sensitive and unbiased in vitro profiling of Cas-dependent base editor genome-wide off-target activity V

Project description:To assess genome-wide off-target activity of base editors and Cas9 nucleases identified by CHANGE-seq-BE, Digenome-seq, and CHANGE-seq, we performed hybrid capture sequencing for five therupatic loci (B2M, CBLB, CD7, CIITA, PDCD1) in human primary T-cells and PCSK9 in human hepatocytes. We observed high on-target editing for ABE, CBE, Cas9 mRNA edited cells and potential off-targets confirmed by hybrid capture sequencing. Our results demonstrate that CHANGE-seq-BE is highly sensitive than Digenome-seq to detect more bona fide off-targets with cellular activity ranging from 0.5% to 92.2%. Moreover, ABE exhibit more off-targets than Cas9 under same delivery conditions.

2025-11-05 | GSE308237 | GEO

CHANGE-seq-BE enables simultaneously sensitive and unbiased in vitro profiling of Cas-dependent base editor genome-wide off-target activity II

Project description:To adapt and apply for cytosone base editors (CBEs), we perfomed CHANGE-seq-BE using eA3A-BE3 to target B2M, CBLB, CD7, CIITA and PDCD1 regions at different loci. CHANGE-seq-BE identified low frequency off-target editing rates ranging from 1.7% to 7.5% that are not identified by Digenome-seq further implies CHANGE-seq-BE is highly sensitive and can identify true off-targets while requiring 20-fold fewer sequencing reads.

2025-11-05 | GSE298534 | GEO

CHANGE-seq-BE enables simultaneously sensitive and unbiased in vitro profiling of Cas-dependent base editor genome-wide off-target activity III

Project description:To assess CHANGE-seq-BE performance, we used it to characterize ABE8e-NRCH base editor activity targeting sickle mutation (HBBS) in the HBB gene and identified additional 29 (53% more) bona fide off-targets than CIRCLE-seq. Furthermore, CHANGE-seq-BE applied for ABE8e targeting five therapeutically relevant loci (B2M, CBLB, CD7, CIITA, and PDCD1) in human primary T-cells and compared with Digenome-seq. CHANGE-seq-BE is highly sensitive and capable of detecting bona fide off-targets while requiring approximately 20-fold less sequencing reads.

2025-11-05 | GSE307740 | GEO

Adenine base editors induce off-target structure variations in mouse embryos and primary human T cells

Project description:Background The safety of CRISPR-based gene editing methods is of the utmost priority in clinical applications. Previous studies have reported that Cas9 cleavage induced frequent aneuploidy in primary human T cells, but whether cleavage-mediated editing of base editors would generate off-target structure variations remains unknown. Here, we investigated the potential off-target structural variations associated with CRISPR/Cas9, ABE and CBE editing in mouse embryos and primary human T cells by whole-genome sequencing and single-cell RNA-seq analyses. Results The results showed that both Cas9 and ABE generated off-target structural variations (SVs) in mouse embryos, while CBE induced rare SVs. In addition, off-target large deletions were detected in 32.74% of primary human T cells transfected with Cas9 and 9.17% of cells transfected with ABE. Moreover, Cas9-induced aneuploid cells activated the P53 and apoptosis pathways, whereas ABE-associated aneuploid cells significantly upregulated cell cycle-related genes and arrested in G0 phase. A percentage of 16.59% and 4.29% aneuploid cells were still observable at 3 weeks post transfection of Cas9 or ABE. These off-target phenomena in ABE were universal as observed in other cell types such as B cells and Huh7. Furthermore, the off-target SVs were significantly reduced in cells treated with high-fidelity ABE (ABE-V106W). Conclusions This study raises urgent need for minimizing the off-target SVs of CRISPR/Cas9 and ABE.

2024-11-01 | GSE280767 | GEO

Quadruple adenine base edited allogeneic CAR T cells outperform CRISPR/Cas9 nuclease engineered T cells [RNA-seq]

Project description:Genome editing technologies have enabled the clinical development of allogeneic cellular therapies, yet the optimal gene editing modality for multiplex editing of therapeutic T cell product manufacturing remains elusive. In this study, we conducted a comprehensive comparison of CRISPR/Cas9 nuclease and adenine base editor (ABE) technologies in generating allogeneic chimeric antigen receptor (CAR) T cells, utilizing extensive in vitro and in vivo analyses. Both methods achieved high editing efficiencies across four target genes, critical for mitigating graft-versus-host disease and allograft rejection: TRAC or CD3E, B2M, CIITA, and PVR. Notably, ABE demonstrated higher manufacturing yields and distinct off-target profiles compared to Cas9, with translocations observed exclusively in Cas9-edited products. Functionally, ABE-edited CAR T cells exhibited superior in vitro effector functions under continuous antigen stimulation, including enhanced proliferative capacity and increased surface CAR expression. Transcriptomic analysis revealed that ABE editing resulted in reduced activation of p53 and DNA damage response pathways at baseline, along with sustained activation of metabolic pathways during antigen stress. Consistently, ATAC-seq data indicated that Cas9-edited, but not ABE-edited, CAR T cells showed enrichment of chromatin accessibility peaks associated with double-strand break repair and DNA damage response pathways. In a preclinical leukemia model, ABE-edited CAR T cells demonstrated improved tumor control and extended overall survival compared to their Cas9-edited counterparts. Collectively, these findings position ABE as a superior choice of Cas9 nucleases for multiplex gene editing in therapeutic T cells.

2025-04-09 | GSE290621 | GEO

Quadruple adenine base edited allogeneic CAR T cells outperform CRISPR/Cas9 nuclease engineered T cells [ATAC-seq]

2025-04-09 | GSE290620 | GEO

Project description:Multiplex Digenome-seq

| PRJNA900689 | ENA

Gene expression profile at the single cell level of wild type and Cbl and Cblb knock-out organoids isolated from mouse intestional crypts.

Project description:Organoid grown from intestinal crypts of Cbl flox/flox; Cbl-bflox/flox; R26Cre ERT2 mice were in presence or absence of 4-hydroxy-tamoxifen to delete Cbl and Cblb. Single cell RNA sequencing were performed in these two conditions to undestand the change in cell populations due to Cbl and Cblb depeletion.

2023-07-01 | GSE236186 | GEO

CRISPR-based adenine editors correct nonsense mutations in a cystic fibrosis organoid biobank.

Project description:Adenine base editing (ABE) enable enzymatic conversion from A-T into G-C base pairs. ABE hold promise for clinical application, as it does not depend on the introduction of double-strand breaks, contrary to conventional CRISPR/Cas9-mediated genome engineering. Here we describe a cystic fibrosis (CF) intestinal organoid biobank, representing 646 patients, of which ~20% can theoretically be repaired by ABE. We apply the SpCas9-ABE (PAM recognition sequence: NGG) and the xCas9-ABE (PAM recognition sequence: NGN) on four selected CF organoid samples. Genetic and functional repair was obtained in all four cases, while whole genome sequencing (WGS) of corrected lines of two patients did not detect off-target mutations. These observations exemplify the value of large, patient-derived organoid biobanks representing hereditary disease, and indicate that ABE may be safely applied in human cells. Whole genome sequencing of Cas9 repaired cystic fibrosis organoids Three SpCas9-ABE (R785X/R785X) and three xCas9-ABE-repaired organoid clones (F508del/R553X) and their respective unrepaired control organoids were paired-end whole genome sequenced using Illumina Novaseq 6000 system. This WGS confirmed the Cas9 repair worked in these organoids and showed no off-target hits nor no mutational hotspots created by the repair system.

| EGAS00001003951 | EGA

CHANGE-seq reveals the genome-wide activity of CRISPR-Cas9 [ChIP-seq]

Project description:We recently developed CHANGE-seq (Circularization for High-throughput Analysis of Nuclease Genome-wide Effects by Sequencing), a fast, streamlined, Tn5 tagmentation-based assay for measuring the genome-wide activity of Cas9 in vitro that is easily scalable to many targets and samples. In this study, we directly compare CIRCLE-seq to CHANGE-seq and systematically evaluate Cas9 genome-wide activity on 110 targets across 13 therapeutically-relevant loci leveraging CHANGE-seq. We validate the sensitivity of CHANGE-seq for identifying sites of bona fide cellular off-target mutations using GUIDE-seq and sensitive targeted tag sequencing. Additionally, we sought to evaluate the impact of chromatin accessibility on cellular off-target activity by comparing CHANGE-seq with matched GUIDE-seq cellular off-target, chromatin, and transcriptional profiles generated from the same human primary T-cells.

2020-06-15 | GSE149295 | GEO

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