Ultra-deep sequencing reveals no evidence of oncogenic mutations or enrichment by ex vivo CRISPR/Cas9 genome editing in human hematopoietic stem and progenitor cells

As CRISPR-based therapies enter the clinic, evaluation of the safety remains a critical and still active area of study. While whole genome sequencing is an unbiased method for identifying somatic mutations introduced by ex vivo culture and genome editing, this methodology is unable to attain sufficient read depth to detect extremely low frequency events that could result in clonal expansion. As a solution, we utilized an exon capture panel to facilitate ultra-deep sequencing of >500 tumor suppressors and oncogenes most frequently altered in human cancer. We used this panel to investigate whether transient delivery of high-fidelity Cas9 protein targeted to three different loci (using guide RNAs (gRNAs) corresponding to sites at AAVS1, HBB, and ZFPM2) at day 4 and day 10 timepoints post-editing resulted in the introduction or enrichment of oncogenic mutations. In three separate primary human HSPC donors, we identified a mean of 1,488 variants per Cas9 treatment (at <0.07% limit of detection). After filtering to remove germline and/or synonymous changes, a mean of 3.3 variants remained per condition, which were further reduced to six total mutations after removing variants in unedited treatments. Of these, four variants resided at the predicted off-target site in the myelodysplasia-associated EZH2 gene that were subject to ZFPM2 gRNA targeting in Donors 2 and 3 at day 4 and day 10 timepoints. While Donor 1 displayed on-target cleavage at ZFPM2, we found no off-target activity at EZH2. Sanger sequencing revealed a homozygous single nucleotide polymorphism (SNP) at position 14bp distal from the Cas9 protospacer adjacent motif in EZH2 that eliminated any detectable off-target activity. We found no evidence of exonic off-target INDELs with either of the AAVS1 or HBB gRNAs. These findings indicate 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 outside the seed region of the gRNA protospacer is sufficient to eliminate Cas9 off-target activity with this method of delivery into primary, repair competent human HSPCs.