Multiplex HDR for Disease and Correction Modeling of SCID by CRISPR Genome Editing in Human HSPCs

Severe combined immunodeficiency (SCID) is a group of monogenic primary immunodeficiencies caused by mutations in genes involved in the process of lymphocyte maturation and function. CRISPR-Cas9 gene editing of the patients own hematopoietic stem and progenitor cells (HSPCs) ex vivo could provide a therapeutic alternative to allogeneic hematopoietic stem cell transplantation, the current gold standard for treatment of SCID patients. Using CRISPR-Cas9/rAAV6 gene-editing, we engineered genotypes in healthy donor (HD)-derived CD34+ HSPCs, thus eliminating the need for rare patient samples, that model both SCID and the therapeutic outcomes of gene-editing therapies for SCID via multiplexed homology directed repair (HDR). Firstly, we developed a SCID disease model via knock-out of both alleles of genes critical to the development of lymphocytes; and secondly, we established a knock-in/knock-out (KI-KO) strategy to develop a proof-of-concept gene correction. Lastly, we performed successful gene correction of RAG2-SCID patient-derived CD34+ HSPCs, highlighted by their successful development into CD3+ T cells with diverse TCR repertoires. By using CRISPR-Cas9, multiplexed HDR, and HD-derived CD34+ HSPCs, we outline an approach for the study of human lymphopoiesis and present a way for researchers to determine the optimal configuration for CRISPR-Cas9 gene correction for other SCIDs and additional recessive blood disorders.