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Controlling DNA-deletion sizes, chromosomal translocations and aneuploidy in CRISPR-edited human T cells by the strength of TCR stimulation and pifithrin-α

ABSTRACT: CRISPR/Cas9-engineered human T cells hold great promise in improving T cell effector functions for adoptive T cell therapies. To ensure safety during CRISPR/Cas9-editing optimized gRNAs, novel variants or prediction tools for indel outcomes have been developed. Still, several reports describe the occurrence of CRISPR-induced chromosomal aberrations, such as loss of large DNA fragments or even aneuploidy. So far, the measures to increase the safety of T cell products focused on the CRISPR Cas9 system. However, T cell-intrinsic features, such as their massive expansion after TCR stimulation, have not been fully taken into consideration. Here, we describe driving forces of indel formation in primary human T cells. Increased T cell activation and proliferation speed correlate with larger deletions. Editing of non-activated T cells reduces the risk of unwanted large deletions but with the downside of reduced knock-out efficiencies. An alternative strategy to reduce the risk of large deletions is the addition of the small molecule pifithrin-α after CRISPR/Cas9 editing. Pifithrin-α treatment results in smaller, more defined deletions and reduces the risk of CRISPR-induced chromosomal translocations and aneuploidy in a p53-independent manner while maintaining the functionality of CRISPR-engineered T cells. Controlling T cell activation and the addition of pifithrin-α are easily accessible strategies for safer CRISPR/Cas9-engineering for adoptive T cell therapies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE249934 | GEO | 2024/09/27

REPOSITORIES: GEO

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Controlling DNA-deletionsizes in CRISPR-edited human T cells by the strength of TCR stimulation and pifithrin-α

Project description:CRISPR/Cas9-engineered human T cells hold great promise in improving T cell effector functions for adoptive T cell therapies. To ensure safety during CRISPR/Cas9-editing optimized gRNAs, novel variants or prediction tools for indel outcomes have been developed. Still, several reports describe the occurrence of chromosomal truncations or loss of large DNA fragments of up to several kbs after CRISPR/Cas9 editing. So far, the measures to increase the safety of T cell products focused on the CRISPR Cas9 system. However, T cell-intrinsic features such as their massive expansion after TCR stimulation have not been taken into consideration. Here, we describe driving forces of indel formation in primary human T cells. Increased T cell activation and proliferation speed correlate with higher knock-out rates and larger deletions. Editing of non-activated T cells reduces the risk of unwanted large deletions but with the downside of reduced knock-out efficiencies. An alternative strategy to reduce the risk of large deletions is the addition of the small molecule pifithrin-α after CRISPR/Cas9 editing. Pifithrin-α treatment results in a smaller, more defined deletion pattern. PFT-a furthermore reduces the risk of unwanted chromosomal translocations and aneuploidy while maintaining the functionality of CRISPR-engineered T cells. Controlling T cell activation and the addition of pifithrin-α are easily accessible strategies for safer CRISPR/Cas9-engineering for adoptive T cell therapies.

2024-09-26 | GSE249194 | GEO

Indel rate analysis:Start codon disruption with CRISPR/Cas9 prevents murine Fuchs' endothelial corneal dystrophy

Project description:A missense mutation of collagen type VIII alpha 2 chain (COL8A2) gene leads to early onset Fuchs’ endothelial corneal dystrophy (FECD), which can cause blindness through progressive loss of corneal endothelial cells. We established a novel procedure for achieving structural and functional rescue of the post-mitotic corneal endothelium without surgery, using CRISPR/Cas9-based postnatal gene editing in a mouse model of FECD. A single intraocular injection of an adenovirus encoding both the Cas9 gene and guide RNA (Ad-Cas9-Col8a2gRNA) at a titer below the cytotoxic threshold, efficiently knocked down COL8A2 protein expression in corneal endothelial cells, prevented endothelial cell loss, and rescued corneal endothelium pumping function in adult Col8a2 mutant mice. Here, to determine the indel rate in mouse corneal endothelium, we performed deep sequencing of PCR products (including the target site) amplified from genomic DNA of corneal endothelium. We found that the indel rate was 23.7 ± 4.5% in mouse corneal endothelium. Most insertions were 1bp insertions (19.8 ± 4.0% in total reads), while 2bp deletions were the most frequent (1.0 ± 0.3% in total reads). We moreover found that A or T insertion was predominant, with the proportion of A:T:G:C being 48.7 : 44.6 : 1.8 : 4.9.

2020-09-01 | GSE146998 | GEO

miRNA-seq of HEK293 cell lines on Illumina iSeq100 instrument

Project description:A validation experiment performed on HEK293 cell lines after genome editing. The design contains three duplicate runs consisted of: HEK293 wild type cell line HEK293 with MIR484 gene knockdown using CRISPR-Cas9 HEK293 with MIR185 gene knockout using CRISPR-Cas9

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CRISPR/Cas9-induced structural variations expand in T lymphocytes in vivo

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

Retroelement insertion in a CRISPR/Cas9 editing site in the early embryo intensifies genetic mosaicism

Project description:Continued CRISPR/Cas9-mediated editing activity that allows differential and asynchronous modification of alleles in successive cell generations expands allelic complexity. To understand the earliest events during CRISPR/Cas9 editing and the allelic selection among the progeny of subsequent cell divisions, we inspected in detail the genotypes of 4- and 8-cell embryos and embryonic stem cells (ESCs) after microinjection of a CRISPR toolkit into the zygotes. We found a higher editing frequency in 8-cell embryos than in 4-cell embryos, indicating that the CRISPR/Cas9 activity persisted through the 8-cell stage. Analysis of a CRISPR/Cas9 transgenic founder mouse revealed that four different alleles were present in its organs in different combinations and that its germline included three different mutant alleles, as shown by the genotypes of the pups. The indel depth, which measured the extent of indels at the sequence level within single embryos, decreased significantly as the embryos advanced to form ESCs, suggesting that exclusion of fatal indels occurred in the subsequent cell generations. Interestingly, we discovered that the CRISPR sites frequently contained introduced retroelement sequences and that this occurred preferentially with certain classes of retroelements. Therefore, in addition to CRISPR/Cas9’s innate mechanism of separate, differential enzymatic modifications of alleles, the frequent retroelement insertions that occur in early mouse embryos during CRISPR/Cas9 editing further expand the allelic diversity and mosaicism in the resulting transgenic founders.

2019-10-22 | GSE136393 | 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

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2022-03-05 | E-MTAB-11470 | biostudies-arrayexpress

Haplotype-phased Callithrix jacchus embryonic stem cell line for genome editing using CRISPR/Cas9

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2018-07-09 | PRJEB27676 | EVA

Targeted DNA-seq of Human HepG2 cells following editing with CRISPR/Cas9 upon chromatin modulation with TSA and EZH2i

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2018-12-13 | E-MTAB-7091 | biostudies-arrayexpress

Using a Dual CRISPR/Cas9 Approach to Gain Insight into the Role of LRP1B in Glioblastoma

Project description:LRP1B remains one of the most altered genes in cancer, although its relevance in cancer biology is still unclear. Recent advances in gene editing techniques, particularly CRISPR/Cas9 systems, offer new opportunities to evaluate the function of large genes, like LRP1B. Using a dual sgRNA CRISPR/Cas9 gene editing approach, this study aimed to assess the impact of disrupting LRP1B in glioblastoma cell biology. Four sgRNAs were designed for the dual targeting of two LRP1B exons (1 and 85). The U87 glioblastoma (GB) cell line was transfected with CRISPR/cas9 PX459 vectors. To assess LRP1B gene alterations and expression, PCR, Sanger DNA sequencing, qRT-PCR were carried out. Three clones (clones B9, E6 and H7) were further evaluated. All clones presented altered cellular morphology, increased cellular and nuclear size and changes in ploidy. Two clones (E6 and H7) showed a significant decrease in cell growth, both in vitro and in the in vivo CAM assay. Proteomic analysis of the clones’ secretome identified differentially ex-pressed proteins, that had not been previously associated with LRP1B alterations. This study demonstrates that the dual sgRNA CRISPR/Cas9 strategy can effectively edit LRP1B in GB cells, providing new insights into the impact of LRP1B deletions in GBM biology.

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