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| Status |
Public on Jan 09, 2018 |
| Title |
Abnormal RNA splicing and genomic instability after induction of DNMT3A mutations by CRISPR/Cas9 gene editing [Exome-Seq] |
| Organism |
Homo sapiens |
| Experiment type |
Other
|
| Summary |
Purpose: DNA methyltransferase 3A (DNMT3A) mediates de novo DNA methylation. Mutations in DNMT3A are associated with hematological malignancies, most frequently acute myeloid leukemia. DNMT3A mutations are hypothesized to establish a pre-leukemic state, rendering cells vulnerable to secondary oncogenic mutations and malignant transformation. However, the mechanisms by which DNMT3A mutations contribute to leukemogenesis are not well-defined.
Methods: Whole-exome sequencing of extracted DNA was performed to confirm DNMT3A mutations. Library construction, exon capture, and sequencing was performed by Otogenetics (Atlanta, GA, USA). In brief, paired-end libraries were generated using the Illumina TruSeq DNA sample preparation kit. Exons were enriched using the Agilent Human All Exon V5 (51 Mb) capture system. Illumina HiSeq2500 was used for sequencing with a paired-end sequencing length of 100-125 bp and approximately 70 million reads per sample.
Results: the DNMT3A mutations induced by K562 cells were transfected with a plasmid encoding Cas9, a CRISPR guide-RNA (gRNA) targeting the DNMT3A gene, and green fluorescent protein (GFP) followed by single-cell sorting. GFP-positive single cell clones were genotyped to confirm the presence of DNMT3A mutations. MT2-MT5 were verified by analysis of whole-exome sequencing data, and WTblk and WT1 were again confirmed to have no mutations in the DNMT3A gene.
Conclusions: CRISPR/Cas9 gene editing allowed the generation of DNMT3A-mutated K562 cells that may be used to model effects of DNMT3A mutations in human cells. Our findings implicate aberrant splicing and induction of genomic instability as potential mechanisms by which DNMT3A mutations might predispose to malignancy.
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| Overall design |
whole-exome sequencing of wild type (WT) and DNMT3A mutated K562 cell lines were generated by deep sequencing using Illumina HiSeq2500
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| Contributor(s) |
Banaszak LG |
| Citation(s) |
29324392 |
| |
| Submission date |
Mar 15, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Valentina Giudice |
| E-mail(s) |
[email protected]
|
| Organization name |
NHLBI
|
| Department |
Hematology Branch
|
| Street address |
10 Center Drive
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20814 |
| Country |
USA |
| |
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| Platforms (1) |
| GPL16791 |
Illumina HiSeq 2500 (Homo sapiens) |
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| Samples (6)
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| This SubSeries is part of SuperSeries: |
| GSE96635 |
Abnormal RNA splicing and genomic instability after induction of DNMT3A mutations by CRISPR/Cas9 gene editing |
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| Relations |
| BioProject |
PRJNA379277 |
| SRA |
SRP101937 |
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