The TRACE-Seq method tracks recombination alleles and identifies clonal reconstitution dynamics of gene targeted human hematopoietic stem cells

Rajiv Sharma,Armon Azizi,Joab Camarena,Matthew H Porteus,Thomas Köhnke,Gang Bao,Yidan Pan,Ravindra Majeti,Daniel P Dever,Ciaran M Lee

doi:10.1038/s41467-020-20792-y

Abstract

Targeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may enable the treatment of genetic diseases of the blood and immune system. It is now possible to correct mutations at high frequencies in HSPCs by combining CRISPR/Cas9 with homologous DNA donors. Because of the precision of gene correction, these approaches preclude clonal tracking of gene-targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a methodology that utilizes barcoded AAV6 donor template libraries, carrying in-frame silent mutations or semi-randomized nucleotides outside the coding region, to track the in vivo lineage contribution of gene-targeted HSPC clones. By targeting the HBB gene with an AAV6 donor template library consisting of ~20,000 possible unique exon 1 in-frame silent mutations, we track the hematopoietic reconstitution of HBB targeted myeloid-skewed, lymphoid-skewed, and balanced multi-lineage repopulating human HSPC clones in mice. We anticipate this methodology could potentially be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings.

Highlights

Targeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may enable the treatment of genetic diseases of the blood and immune system
TRACE-Seq improves the understanding of the clonal dynamics of hematopoietic stem and progenitor cells following homologous recombination-based genome editing using two different gene targets (HBB and AAVS1)
The data demonstrate that Cas9/synthesized guide RNA (sgRNA) and associated virus serotype 6 (AAV6) gene editing targets four distinct types of hematopoietic cells capable of engraftment, including: (1) rare and potent hematopoietic balanced long-term hematopoietic stem cell (LT-HSC), (2) rare lymphoid skewed progenitors, (3) rare and potent myeloid skewed progenitors, and (4) more common and less proliferative myeloid skewed HSPCs

Summary

Introduction

Targeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may enable the treatment of genetic diseases of the blood and immune system. By targeting the HBB gene with an AAV6 donor template library consisting of ~20,000 possible unique exon 1 in-frame silent mutations, we track the hematopoietic reconstitution of HBB targeted myeloid-skewed, lymphoidskewed, and balanced multi-lineage repopulating human HSPC clones in mice We anticipate this methodology could potentially be used for HSPC clonal tracking of Cas[9] RNP and AAV6mediated gene targeting outcomes in translational and basic research settings. While no severe adverse events have been reported resulting from insertional mutagenesis in more than 200 patients transplanted with LV ex vivo manipulated HSPCs2, efficacy in restoring protein/cell function and disease amelioration has varied In some diseases, this lack of therapeutic efficacy is possibly the result of irregular spatiotemporal transgene expression due to the semi-random integration patterns of LVs. Tracking the transgene integration sites (IS) by deep sequencing has been used to “barcode” clones in heterogeneous cell populations that contribute to blood reconstitution in the human transplantation setting. Genome editing provides an alternative approach to lentiviral integrations to perform permanent genetic engineering of cells

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Conclusion