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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. Daniel P. Dever & Rasmus O. Bak & Andreas Reinisch & Joab Camarena & Gabriel Washington & Carmencita E. Nicolas & Mara Pavel-Dinu & Nivi Saxena & Alec B. Wilkens & Sruthi Mantri & Nobuko Uchida & Ayal, 2016. "CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells," Nature, Nature, vol. 539(7629), pages 384-389, November.
    2. Konrad J. Karczewski & Laurent C. Francioli & Grace Tiao & Beryl B. Cummings & Jessica Alföldi & Qingbo Wang & Ryan L. Collins & Kristen M. Laricchia & Andrea Ganna & Daniel P. Birnbaum & Laura D. Gau, 2020. "The mutational constraint spectrum quantified from variation in 141,456 humans," Nature, Nature, vol. 581(7809), pages 434-443, May.
    3. Grégoire Cullot & Julian Boutin & Jérôme Toutain & Florence Prat & Perrine Pennamen & Caroline Rooryck & Martin Teichmann & Emilie Rousseau & Isabelle Lamrissi-Garcia & Véronique Guyonnet-Duperat & Al, 2019. "CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations," Nature Communications, Nature, vol. 10(1), pages 1-14, December.
    4. Emmanouela Tsagkaraki & Sarah M. Nicoloro & Tiffany DeSouza & Javier Solivan-Rivera & Anand Desai & Lawrence M. Lifshitz & Yuefei Shen & Mark Kelly & Adilson Guilherme & Felipe Henriques & Nadia Amran, 2021. "CRISPR-enhanced human adipocyte browning as cell therapy for metabolic disease," Nature Communications, Nature, vol. 12(1), pages 1-17, December.
    5. Verena Passerini & Efrat Ozeri-Galai & Mirjam S. de Pagter & Neysan Donnelly & Sarah Schmalbrock & Wigard P. Kloosterman & Batsheva Kerem & Zuzana Storchová, 2016. "The presence of extra chromosomes leads to genomic instability," Nature Communications, Nature, vol. 7(1), pages 1-12, April.
    6. Amanda Finck & Saar I. Gill & Carl H. June, 2020. "Cancer immunotherapy comes of age and looks for maturity," Nature Communications, Nature, vol. 11(1), pages 1-4, December.
    7. J. Boutin & J. Rosier & D. Cappellen & F. Prat & J. Toutain & P. Pennamen & J. Bouron & C. Rooryck & J. P. Merlio & I. Lamrissi-Garcia & G. Cullot & S. Amintas & V. Guyonnet-Duperat & C. Ged & J. M. B, 2021. "CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells," Nature Communications, Nature, vol. 12(1), pages 1-12, December.
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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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