PT  - JOURNAL ARTICLE
AU  - Rajiv Sharma
AU  - Daniel P Dever
AU  - Ciaran M Lee
AU  - Armon Azizi
AU  - Yidan Pan
AU  - Joab Camarena
AU  - Thomas Köhnke
AU  - Gang Bao
AU  - Matthew H Porteus
AU  - Ravindra Majeti
TI  - TRACE-Seq Reveals Clonal Reconstitution Dynamics of Gene Targeted Human Hematopoietic Stem Cells
AID  - 10.1101/2020.05.25.115329
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.05.25.115329
4099  - http://biorxiv.org/content/early/2020/05/28/2020.05.25.115329.short
4100  - http://biorxiv.org/content/early/2020/05/28/2020.05.25.115329.full
AB  - Targeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may usher in a new class of medicines to treat genetic diseases of the blood and immune system. With state-of-the-art methodologies, it is now possible to correct disease-causing mutations at high frequencies in HSPCs by combining ribonucleoprotein (RNP) delivery of Cas9 and chemically modified sgRNAs with homologous DNA donors via recombinant adeno-associated viral vector serotype six (AAV6). However, because of the precise nucleotide-resolution nature of gene correction, these current approaches do not allow for clonal tracking of gene targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a novel methodology that utilizes barcoded AAV6 donor template libraries, carrying either in-frame silent mutations or semi-randomized nucleotide sequences 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 immunodeficient mice. We anticipate that this methodology has the potential to be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings.Competing Interest StatementM.H.P. has equity and serves on the scientific advisory board of CRISPR Therapeutics and Allogene Therapeutics. R.M. is a founder, consultant, equity holder, and serves on the Board of Directors of Forty Seven Inc. However, none of these companies had input into the design, execution, interpretation, or publication of the work in this manuscript.