COMBINED LENTIVIRAL- AND RNA-MEDIATED CRISPR/CAS9 DELIVERY FOR EFFICIENT AND TRACEABLE GENE EDITING IN HUMAN HEMATOPOIETIC STEM AND PROGENITOR CELLS

Abstract

The CRISPR/Cas9 system has been highlighted as an ideal tool for large-scale forward genetic screens in mammalian cells. To deliver CRISPR libraries by lentiviral transduction is advantageous in a pooled screen context where an integrated and traceable component is required. However, lentiviral delivery of the CRISPR components to primary human hematopoietic stem and progenitor cells (HSPCs) has proven challenging and limited the screening opportunities in these cells. Here, we addressed these challenges by developing a hybrid system combining stable delivery of single guide RNA (sgRNA) by lentiviral transduction, with transient delivery of Cas9 by electroporation. We optimized and compared delivery modes of Cas9 in cord blood-derived CD34+ HSPCs transduced with lentiviral sgRNAs targeting the gene PTPRC (CD45), and delivered Cas9 either as mRNA or protein by electroporation to assess gene knockout in terms of loss of CD45 expression evaluated by flow cytometry. We found that Cas9 mRNA is preferable to Cas9 protein in terms of editing efficiency and acceptable cytotoxicity. We further implemented an optimized sgRNA structure, which significantly improved the editing efficiency, reaching 90% knockout of CD45 in GFP+ sgRNA transduced cells. Importantly, this was also observed for several sgRNAs targeting CD29 and CD44, reaching editing efficiencies such as 62% and 88% compared to 30% and 76%, respectively. This gene editing did not affect CD34 expression or in vitro colony forming capacity compared to untreated or mock treated cells. Furthermore, transplantation experiments in immunodeficient mice showed that the gene edited HSPCs retained efficient reconstitution capacity in vivo. Taken together, this hybrid system not only balances efficient editing with low cytotoxicity, but also enables cost-effective, traceable gene editing for screening purposes in human HSPCs.