Abstract
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-mediated genome editing holds remarkable promise for the treatment of human genetic diseases. However, the possibility of off-target Cas9 activity remains a concern. To address this issue using clinically relevant target cells, we electroporated Cas9 ribonucleoprotein (RNP) complexes (independently targeted to two different genomic loci, the CXCR4 locus on chromosome 2 and the AAVS1 locus on chromosome 19) into human mobilized peripheral blood-derived hematopoietic stem and progenitor cells (HSPCs) and assessed the acquisition of somatic mutations in an unbiased, genome-wide manner via whole genome sequencing (WGS) of single-cell-derived HSPC clones. Bioinformatic analysis identified >20,000 total somatic variants (indels, single nucleotide variants, and structural variants) distributed among Cas9-treated and non-Cas9-treated control HSPC clones. Statistical analysis revealed no significant difference in the number of novel non-targeted indels among the samples. Moreover, data analysis showed no evidence of Cas9-mediated indel formation at 623 predicted off-target sites. The median number of novel single nucleotide variants was slightly elevated in Cas9 RNP-recipient sample groups compared to baseline, but did not reach statistical significance. Structural variants were rare and demonstrated no clear causal connection to Cas9-mediated gene editing procedures. We find that the collective somatic mutational burden observed within Cas9 RNP-edited human HSPC clones is indistinguishable from naturally occurring levels of background genetic heterogeneity.
Highlights
Hematopoietic stem and progenitor cells (HSPCs) are important targets of gene-based regenerative therapies [1]
To address the significance of off-target CRISPR/CRISPR-associated protein 9 (Cas9) activity in human blood stem cells, we performed Cas9 RNP-based genome editing in peripheral blood-mobilized human hematopoietic stem and progenitor cells (HSPCs) obtained from a healthy donor and characterized the genome-wide accumulation of post-editing somatic mutations using high-throughput whole genome sequencing (WGS) analysis of single-cell-derived HSPC clones (Figure 1)
CRISPR/Cas9 activity in primary, human HSPCs independently edited at two different genomic loci
Summary
Hematopoietic stem and progenitor cells (HSPCs) are important targets of gene-based regenerative therapies [1]. Permanent correction of inherited hematologic, metabolic and immunologic disorders. Genes 2020, 11, 1501 can be achieved owing to the remarkable ability of long-term repopulating HSPCs to reconstitute and maintain a functional hematopoietic system in vivo. Unequivocal clinical benefits have been obtained over the past decade by vector-mediated addition of a therapeutic gene to HSPCs using replication-defective integrating retroviruses [2]. Transformative technological advances have allowed the precise editing of cellular genomes, potentially obviating concerns regarding insertional mutagenesis inevitably associated with integrating vectors, as well as enabling appropriate transgene expression levels from endogenous cellular promoters and extending gene therapies to disorders requiring genetic correction of abnormal gene products rather than simple gene addition [2,3,4].
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