High-throughput amplicon sequencing were performed on CD34+ hematopoietic stem and progenitor cells (HSPCs) derived from 4 healthy donors and one unaffected heterozygous carrier of a CYBB c.252G>A variant causing X-linked granulomatous disease. Targeted genome editing were performed in the HSPCs of CYBA and CYBB genes using CRISPR-Cas9 and recombinant AAV6. To assess gene editing outcomes in CD34+ HSPCs, the targeted loci were amplified from genomic DNA using a three-step PCR protocol. Briefly, the first PCR (PCR1) amplified the genomic region of interest with at least one primer binding outside the homology arms of the HDR templates. The second PCR (PCR2) attached Illumina TruSeq sequencing adapters. The third PCR (PCR3) added indexes along with the P5 and P7 sequence adapters. PCR1 was run on either 5 µl QuickExtract lysate or 660 ng purified gDNA in a 50 µL Phusion Plus reaction for 28 cycles and purified by excision from a 1% agarose gel (Omega Bio-Tek) to ensure removal of residual DNA repair template. PCR2 was run on 10 µL purified PCR1 product in a 25 µL Phusion Plus reaction for 8 cycles. PCR3 was run on 4 ng purified PCR2 product for 8 cycles. Sequence-ready amplicons were then sequenced with 150-bp paired-end reads on an Illumina iSeq 100 or MiniSeq. Editing rates were determined with CRISPResso2 in HDR mode using standard parameters. The dataaset here contains paired raw fastq files of each sample.
| Samples | Technology | Sequencing platform |
|---|---|---|
| Mock_1_L001_R1_001.fastq.gz, Mock_1_L001_R2_001.fastq.gz, RNP_AAV_1_L001_R1_001.fastq.gz, RNP_AAV_1_L001_R2_001.fastq.gz, …,RNP_AAV_3_L001_R1_001.fastq.gz, RNP_AAV_3_L001_R2_001.fastq.gz | Illumina NGS | Illumina iSeq100 |
Wolff et al. Targeted gene editing and near-universal cDNA insertion of CYBA and CYBB as a treatment for chronic granulomatous disease.
External researchers (academic or commercial) interested in analyzing sequencing datasets on patient stem cells will need to contact the Data Access Committee via email to giehm@biomed.au.dk. The Data Access Committee consists of Jacob Giehm Mikkelsen, Rasmus O. Bak (both at Department of Biomedicine, Aarhus University) and Trine Mogensen, Mette Holm, and Bjarne Kuno Møller (all at Department of Clinical Medicine, Aarhus University). Due to Danish Law, for the authors to be allowed to share the data (pseudonymized) it will require prior approval from The Danish National Committee on Health Research Ethics (or similar) for the specific new research goal. The author (based in Denmark) has to submit the application for ethical approval, with the external researcher(s) as named collaborator(s). In addition to ethical approval, a Collaboration Agreement and a Data Processing Agreement is required, both of which must be approved by the legal office of the institution of the author (data owner) and the legal office of the institution of the external researcher (data processor)