CRISPR/Cas9-induced structural variations expand in T lymphocytes in vivo
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ABSTRACT:
PROVIDER: PRJNA837595 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR19179004_1.fastq.gz | Fastqsanger.gz | |||
| SRR19179004_2.fastq.gz | Fastqsanger.gz | |||
| SRR19179005_1.fastq.gz | Fastqsanger.gz | |||
| SRR19179005_2.fastq.gz | Fastqsanger.gz | |||
| SRR19179006_1.fastq.gz | Fastqsanger.gz |
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Project description:CRISPR/Cas9 has been adapted to disrupt endogenous genes in adoptive T-lymphocyte therapy to prevent graft-versus-host disease. However, genome editing also generates prevalent deleterious structural variations (SVs), including chromosomal translocations and large deletions, raising safety concerns about reinfused T cells. Here, we dynamically monitored the progression of SVs in a mouse model of T-cell receptor (TCR)-transgenic T-cell adoptive transfer, mimicking TCR T therapeutics. Remarkably, CRISPR/Cas9-induced SVs persist and undergo clonal expansion in vivo after three weeks or even two months, evidenced by high enrichment and low junctional diversity of identified SVs post infusion. Specifically, we detected 128 expanded translocations, with 20,615 as the highest number of amplicons. The identified SVs are stochastically selected among different individuals and show an inconspicuous locus preference. Moreover, viral DNA integrations are routinely detected in edited T cells and also undergo clonal expansion as SVs do. The persistent SVs and viral DNA integrations in the infused T cells may constantly threaten genome integrity, drawing immediate attention to the safety of CRISPR/Cas9-engineered T cells mediated immunotherapy.
2022-10-12 | GSE202887 | GEO
Project description:CRISPR/Cas9 has been adapted to disrupt endogenous genes in adoptive T-lymphocyte therapy to prevent graft-versus-host disease. However, genome editing also generates prevalent deleterious structural variations (SVs), including chromosomal translocations and large deletions, raising safety concerns about reinfused T cells. Here, we dynamically monitored the progression of SVs in a mouse model of T-cell receptor (TCR)-transgenic T-cell adoptive transfer, mimicking TCR T therapeutics. Remarkably, CRISPR/Cas9-induced SVs persist and undergo clonal expansion in vivo after three weeks or even two months, evidenced by high enrichment and low junctional diversity of identified SVs post infusion. Specifically, we detected 128 expanded translocations, with 20 615 as the highest number of amplicons. The identified SVs are stochastically selected among different individuals and show an inconspicuous locus preference. Similar to SVs, viral DNA integrations are routinely detected in edited T cells and also undergo clonal expansion. The persistent SVs and viral DNA integrations in the infused T cells may constantly threaten genome integrity, drawing immediate attention to the safety of CRISPR/Cas9-engineered T cells mediated immunotherapy.
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Project description:Quantifying epigenotype variations between mouse B and CD4+ T lymphocytes
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Project description:Quantifying epigenotype variations between mouse B and CD4+ T lymphocytes
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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.
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