Abstract
Precise and efficient manipulation of genes is crucial for understanding the molecular mechanisms that govern human hematopoiesis and for developing novel therapies for diseases of the blood and immune system. Current methods do not enable precise engineering of complex genotypes that can be easily tracked in a mixed population of cells. We describe a method to multiplex homologous recombination (HR) in human hematopoietic stem and progenitor cells and primary human T cells by combining rAAV6 donor delivery and the CRISPR/Cas9 system delivered as ribonucleoproteins (RNPs). In addition, the use of reporter genes allows FACS-purification and tracking of cells that have had multiple alleles or loci modified by HR. We believe this method will enable broad applications not only to the study of human hematopoietic gene function and networks, but also to perform sophisticated synthetic biology to develop innovative engineered stem cell-based therapeutics.
Highlights
The current gold standard method for studying human hematopoietic stem and progenitor cell (HSPC) gene function has been either overexpression or RNAi-mediated knockdown of genes using lentiviral vectors (Doulatov et al, 2012; Chan et al, 2015)
We and others have previously shown that homologous recombination (HR) in human HSPCs can be efficiently induced by sitespecific nucleases in combination with homologous donor DNA delivered as single-stranded oligonucleotides, integration-defective lentiviral vectors (IDLVs), or by recombinant adeno-associated virus serotype 6 vectors (Dever et al, 2016; DeWitt et al, 2016; De Ravin et al, 2017; Wang et al, 2015; Hoban et al, 2016)
After successful on-target integration of a reporter transgene, FACS-based sorting of transgene reporterhigh-expressing HSPCs was used to purify an HSPC population with >90% targeted integration that displayed long-term repopulation capacity in NOD scid gamma (NSG) mice (Dever et al, 2016). To extend this method beyond the HBB locus for therapeutic genome editing approaches of hemoglobinopathies, we tested six additional loci for their potential to be modified through HR by CRISPR/Cas9 in combination with AAV6-derived donor delivery. These genes are associated with hematopoiesis, hematopoietic malignancies, or safe harbor sites and include: interleukin-2 receptor gamma chain (IL2RG), chemokine (C-C motif) receptor 5 (CCR5), runt-related transcription factor one isoform c (RUNX1c), additional sex combs like 1 (ASXL1), stromal antigen 2 (STAG2), and adeno-associated virus integration site 1 (AAVS1) (Tebas et al, 2014; Genovese et al, 2014; Patel et al, 2012; Mazumdar et al, 2015; Kotin et al, 1992)
Summary
The current gold standard method for studying human hematopoietic stem and progenitor cell (HSPC) gene function has been either overexpression or RNAi-mediated knockdown of genes using lentiviral vectors (Doulatov et al, 2012; Chan et al, 2015). Programmable nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas have been utilized to disrupt genes by the introduction of site-specific DNA double strand breaks (DSBs) that are corrected through non-homologous end-joining (NHEJ) (Hendel et al, 2015; Holt et al, 2010; Saydaminova et al, 2015; Mandal et al, 2014; Schumann et al, 2015; Kim et al, 2014; Lin et al, 2014) This error-prone system creates a heterogeneous mixture of cells with various genotypes of SNPs and small insertions or deletions (INDELs); not all of the genetic changes from INDELs cause functional gene disruption as they may preserve the open reading frame and may not change amino acids essential for protein functions (Shi et al, 2015; Hultquist et al, 2016).
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