Multiplex CRISPR/Cas9-Based Genome Editing in Human Hematopoietic Stem Cells Models Clonal Hematopoiesis and Myeloid Neoplasia

Abstract

SUMMARY Hematologic malignancies are driven by combinations of genetic lesions that have been difficult to model in human cells. We used CRISPR/Cas9 genome engineering of primary adult and umbilical cord blood CD34+ human hematopoietic stem and progenitor cells (HSPCs), the cells of origin for myeloid pre-malignant and malignant diseases, followed by transplantation into immunodeficient mice, to generate genetic models of clonal hematopoiesis and neoplasia. Human hematopoietic cells bearing mutations in combinations of genes observed in myeloid malignancies, including cohesin complex genes, generated immunophenotypically defined neoplastic clones capable of long-term, multi-lineage reconstitution and serial transplantation. Employing these models to investigate therapeutic efficacy, we found that TET2 and cohesin-mutated hematopoietic cells were sensitive to azacitidine treatment. These findings demonstrate the potential for generating genetically-defined models of human myeloid diseases and are suitable for examining the biological consequences of somatic mutations and the testing of therapeutic agents.

Double Selection Enhances the Efficiency of Target-AID and Cas9-Based Genome Editing in Yeast

“…The observation that multiplex genome editing that does not rely on HDR integration of donor DNA causes chromosomal rearrangements at relatively high frequency is worrisome because many recent multiplex mutagenesis workflow rely on PCR based deep sequencing of the target allele to measure success rates ( i.e. , CRISPR-Seq ( Tothova et al 2017 )) of genome editing at multiple loci. This method cannot detect chromosomal rearrangements, and as such the events may have gone unnoticed in many experiments so far.…”

Functional Profiling of Single CRISPR/Cas9-Edited Human Long-Term Hematopoietic Stem Cells

“…Pre-leukemic LT-HSCs are a source of clonal evolution within blood malignancies and can act as a reservoir of relapse after chemotherapy treatment 7 . Thus, modeling and understanding the genetic complexity and cellular heterogeneity seen in human hematological malignancies requires novel methodologies that allow genome editing in LT-HSCs and their functional read-out 3,4 .Recently several studies have demonstrated efficient gene editing of bulk CD34 + populations that are enriched for human HSPCs [8][9][10][11][12][13][14][15][16] . Highly efficient non-homologous end joining (NHEJ) mediated gene disruption of up to 80-90% efficiency has been reported in CD34 + HSPCs 10,11,14,16 .…”

Clonal expansion and myeloid leukemia progression modeled by multiplex gene editing of murine hematopoietic progenitor cells

“…Additionally, a recent study described the generation of CHIP and myeloid disease models by multiplex gene editing of human CD34 + HSPCs using transient plasmid transfection (Tothova et al, 2017). In this model, myeloid malignancies showing expansion of immature myeloid cells or histiocytes emerged when multiplex gene editing was combined with the introduction of oncogenic FLT3-ITD or NPM1 mutants.…”