HEK293T Cells Are Heterozygous for CCR5 Delta 32 Mutation

Qi, Chunxia; Jia, Xiaoxu; Lu, Lingling; Ma, Ping; Wei, Min

doi:10.1371/journal.pone.0152975

Cited by 4 publications

(4 citation statements)

References 28 publications

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“…2D). Surprisingly, the sequencing results also demonstrated all the clones had natural 32-bp deletion on the other allele, which is consistent with the recent report that HEK293T cells are heterozygous for CCR5 D32 mutation [21].…”

Section: Resultssupporting

confidence: 91%

“…Surprisingly, we detected no biallelic integration at first and further sequencing results showed all the clones had the 32‐bp deletion on the other allele. This accidental discovery is just confirmed by the later report that HEK293T cells are heterozygous for CCR5 Δ32 mutation . As the CCR5 .sgRNA target site was chosen from the missing sequence, CCR5 .sgRNA‐Cas9 failed to target the allele with the Δ32 mutation to mediate the integration.…”

Section: Discussionmentioning

confidence: 90%

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Efficient SSA‐mediated precise genome editing using CRISPR/Cas9

et al. 2018

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CRISPR/Cas9 has been emerging as a main player in genome editing field since its advent. However, CRISPR/Cas9-induced precise gene editing remains challenging since it requires no scar left after editing. Among the few reports regarding two-step 'pop in & out' technologies for precise gene editing, the combination of CRISPR/Cas9 with Cre/LoxP demonstrates a higher efficiency, but leaves behind a 34-base pair of tag sequence due to its inherent property. Another method utilizes piggyBac transposon for removing the selection cassette, and its disadvantage is the difficulty in controlling its random reintegration after releasing. Here, we report a novel two-step precise gene-editing method by leveraging the SSA-mediated repair mechanism into the CRISPR/Cas9-mediated gene-editing system. An integrating cassette was developed with positive and negative selection markers, which was flanked by direct repeat sequences with desired mutations as SSA arms. After the targeted integration of the cassette mediated by CRISPR/Cas9-induced homologous-directed repair, cell clones were first selected through the positive selection. In the second round targeting, the selection cassette was removed by the SSA-mediated DNA double-strand break (DSB) repair without any scar left behind. The novel seamless genome editing technique was tested on CCR5 and APP loci, and finally demonstrated, respectively, up to 45.83% and 68% of precise genome editing efficiency. This study provides a new efficient approach for precise genome editing and gene correction.

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Section: Resultssupporting

confidence: 91%

Section: Discussionmentioning

confidence: 90%

Efficient SSA‐mediated precise genome editing using CRISPR/Cas9

et al. 2018

View full text Add to dashboard Cite

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“…3a, b, d, e ), though we detected a lower number of L1-ORFeus insertions compared to MYC and RAG1 loci, likely due to the lower efficiency of the CCR5 CRISPR/Cas9 (Supplementary Fig. 3c ) as well as only one copy of the CCR5 gene can be edited since HEK293T cells are heterozygous for CCR5 delta 32 mutation 57 . Again, retrotransposition of the spliced L1-ORFeus transcript were observed at CCR5 CRIPSR/Cas9 editing site only in L1-ORFeus, but not in L1-RTm (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 82%

Frequency and mechanisms of LINE-1 retrotransposon insertions at CRISPR/Cas9 sites

et al. 2022

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CRISPR/Cas9-based genome editing has revolutionized experimental molecular biology and entered the clinical world for targeted gene therapy. Identifying DNA modifications occurring at CRISPR/Cas9 target sites is critical to determine efficiency and safety of editing tools. Here we show that insertions of LINE-1 (L1) retrotransposons can occur frequently at CRISPR/Cas9 editing sites. Together with PolyA-seq and an improved amplicon sequencing, we characterize more than 2500 de novo L1 insertions at multiple CRISPR/Cas9 editing sites in HEK293T, HeLa and U2OS cells. These L1 retrotransposition events exploit CRISPR/Cas9-induced DSB formation and require L1 RT activity. Importantly, de novo L1 insertions are rare during genome editing by prime editors (PE), cytidine or adenine base editors (CBE or ABE), consistent with their reduced DSB formation. These data demonstrate that insertions of retrotransposons might be a potential outcome of CRISPR/Cas9 genome editing and provide further evidence on the safety of different CRISPR-based editing tools.

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“…Unexpectedly, three fragments were observed in the sorted blasts and LSC from TARP-high pedAML patients and the MV4; 11 cell line. Cloning and sequencing of each fragment (Online Supplementary Figure S5B) revealed that the largest fragments were artificial heteroduplexes, 44 whereas the smallest fragments were identical to the fragments from Kg-1a and LNCaP. Medium-sized fragments were consistently 48 bp longer, and showed the same size as the HSB-2 amplicon, a T-cell acute lymphoblastic leukemia (T-ALL) cell line with functional TRGC2 rearrangements.…”

Section: A B Cmentioning

confidence: 99%

TARP is an immunotherapeutic target in acute myeloid leukemia expressed in the leukemic stem cell compartment

et al. 2019

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I mmunotherapeutic strategies targeting the rare leukemic stem cell compartment might provide salvage to the high relapse rates currently observed in acute myeloid leukemia (AML). We applied gene expression profiling for comparison of leukemic blasts and leukemic stem cells with their normal counterparts. Here, we show that the T-cell receptor γ chain alternate reading frame protein (TARP) is over-expressed in de novo pediatric (n=13) and adult (n=17) AML sorted leukemic stem cells and blasts compared to hematopoietic stem cells and normal myeloblasts (15 healthy controls). Moreover, TARP expression was significantly associated with a fmslike tyrosine kinase receptor-3 internal tandem duplication in pediatric AML. TARP overexpression was confirmed in AML cell lines (n=9), and was found to be absent in B-cell acute lymphocytic leukemia (n=5) and chronic myeloid leukemia (n=1). Sequencing revealed that both a classical TARP transcript, as described in breast and prostate adenocarcinoma, and an AML-specific alternative TARP transcript, were present. Protein expression levels mostly matched transcript levels. TARP was shown to reside in the cytoplasmic compartment and showed sporadic endoplasmic reticulum co-localization. TARP-T-cell receptor engineered cytotoxic T-cells in vitro killed AML cell lines and patient leukemic cells co-expressing TARP and HLA-A*0201. In conclusion, TARP qualifies as a relevant target for immunotherapeutic T-cell therapy in AML.

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HEK293T Cells Are Heterozygous for CCR5 Delta 32 Mutation

Cited by 4 publications

References 28 publications

Efficient SSA‐mediated precise genome editing using CRISPR/Cas9

Efficient SSA‐mediated precise genome editing using CRISPR/Cas9

Frequency and mechanisms of LINE-1 retrotransposon insertions at CRISPR/Cas9 sites

TARP is an immunotherapeutic target in acute myeloid leukemia expressed in the leukemic stem cell compartment

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