Multiplex HDR for Modeling and Correction of RAG2-SCID by CRISPR-Genome Editing in Hematopoietic stem and progenitor cells

Severe combined immunodeficiencies (SCIDs) are a set of life-threatening genetic diseases in which patients are born with mutations in one of more than twenty known genes and are unable to develop functional immune systems. While allogeneic bone marrow transplantation can be curative for these diseases, there remain significant limitations to this approach. In contrast to using viral vectors to deliver transgenes in an uncontrolled fashion, we are working towards developing CRISPR genome editing to correct the RAG2 disease-causing mutations by precisely modifying the genome. Here we first developed a RAG2-SCID disease model using CRISPR-Cas9 and rAAV6 gene-editing through biallelic knock-out in healthy donor (HD)-derived CD34+ HSPCs. Additionally, we established a knock-in/knock-out (KI-KO) strategy to develop a proof-of-concept gene correction in HD CD34+ HSPCs. Lastly, we show successful gene correction of RAG2-SCID patient-derived CD34+ HSPCs which developed into CD3+ T cells with diverse TCR repertoires. Our system outlines an approach for the study of human lymphopoiesis and will allow researchers to determine the optimal configuration for CRISPR-Cas9 gene correction for SCIDs and additional recessive primary immunodeficiency and blood disorders.