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

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 and Matthew H. Porteus ()
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M. Kyle Cromer: University of California, San Francisco
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

Nature Communications, 2022, vol. 13, issue 1, 1-11

Abstract: 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.

Date: 2022
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DOI: 10.1038/s41467-022-32233-z

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