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Clonal Tracking Uncovers Barriers and Validate New Strategies to Enhance Gene Editing in Human Hematopoietic Stem Cells [BAR-seq]

ABSTRACT: By applying a barcoding strategy to clonal tracking of edited cells (BAR-seq), we show that p53 activation triggered by HDR editing significantly shrink the HSPC clonal repertoire in hematochimeric mice, despite engrafted edited clones preserved multilineage and self-renewing capacity. Transient inhibition of p53 restored polyclonal graft composition. We then overcame HSC constraints to HDR by forcing cell cycle progression and upregulating components of the HDR machinery through transient expression of Adenovirus 5 E4orf6/7 protein, which operates the major cell cycle controller E2F. Combined E4orf6/7 expression and p53 inhibition resulted in high and stable HDR editing efficiencies in the human graft, without perturbing repopulation and self-renewing properties of edited HSCs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE144340 | GEO | 2020/04/22

REPOSITORIES: GEO

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Clonal Tracking Uncovers Barriers and Validate New Strategies to Enhance Gene Editing in Human Hematopoietic Stem Cells [RNA-seq]

Project description:By applying a barcoding strategy to clonal tracking of edited cells (BAR-seq), we show that p53 activation triggered by HDR editing significantly shrink the HSPC clonal repertoire in hematochimeric mice, despite engrafted edited clones preserved multilineage and self-renewing capacity. Transient inhibition of p53 restored polyclonal graft composition. We then overcame HSC constraints to HDR by forcing cell cycle progression and upregulating components of the HDR machinery through transient expression of Adenovirus 5 E4orf6/7 protein, which operates the major cell cycle controller E2F. Combined E4orf6/7 expression and p53 inhibition resulted in high and stable HDR editing efficiencies in the human graft, without perturbing repopulation and self-renewing properties of edited HSCs.

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Transient Inhibition of 53BP1 Increases the Frequency of Targeted Integration in Human Hematopoietic Stem and Progenitor Cells

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