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Ultra-deep sequencing validates safety of CRISPR/Cas9 genome editing in human hematopoietic stem and progenitor cells

Author

Listed:
  • M. Kyle Cromer

    (University of California, San Francisco
    Stanford University)

  • Valentin V. Barsan

    (Stanford University)

  • Erich Jaeger

    (Illumina)

  • Mengchi Wang

    (Illumina)

  • Jessica P. Hampton

    (Stanford University)

  • Feng Chen

    (Illumina)

  • Drew Kennedy

    (Illumina)

  • Jenny Xiao

    (Illumina)

  • Irina Khrebtukova

    (Illumina)

  • Ana Granat

    (Illumina)

  • Tiffany Truong

    (Illumina)

  • Matthew H. Porteus

    (Stanford University)

Abstract

As CRISPR-based therapies enter the clinic, evaluation of safety remains a critical and active area of study. Here, we employ a clinical next generation sequencing (NGS) workflow to achieve high sequencing depth and detect ultra-low frequency variants across exons of genes associated with cancer, all exons, and genome wide. In three separate primary human hematopoietic stem and progenitor cell (HSPC) donors assessed in technical triplicates, we electroporated high-fidelity Cas9 protein targeted to three loci (AAVS1, HBB, and ZFPM2) and harvested genomic DNA at days 4 and 10. Our results demonstrate that clinically relevant delivery of high-fidelity Cas9 to primary HSPCs and ex vivo culture up to 10 days does not introduce or enrich for tumorigenic variants and that even a single SNP in a gRNA spacer sequence is sufficient to eliminate Cas9 off-target activity in primary, repair-competent human HSPCs.

Suggested Citation

  • M. Kyle Cromer & Valentin V. Barsan & Erich Jaeger & Mengchi Wang & Jessica P. Hampton & Feng Chen & Drew Kennedy & Jenny Xiao & Irina Khrebtukova & Ana Granat & Tiffany Truong & Matthew H. Porteus, 2022. "Ultra-deep sequencing validates safety of CRISPR/Cas9 genome editing in human hematopoietic stem and progenitor cells," Nature Communications, Nature, vol. 13(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-32233-z
    DOI: 10.1038/s41467-022-32233-z
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    1. Hsiu-Hui Tsai & Hsiao-Jung Kao & Ming-Wei Kuo & Chin-Hsien Lin & Chun-Min Chang & Yi-Yin Chen & Hsiao-Huei Chen & Pui-Yan Kwok & Alice L. Yu & John Yu, 2023. "Whole genomic analysis reveals atypical non-homologous off-target large structural variants induced by CRISPR-Cas9-mediated genome editing," Nature Communications, Nature, vol. 14(1), pages 1-9, December.

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