CRISPR Gene Correction for Severe Combined Immunodeficiency Caused by Mutations in Recombination-Activating Gene 1 and 2 (RAG1 and RAG2)

Genome editing of human hematopoietic stem and progenitor cells (HSPCs) holds a great therapeutic potential. In contrast to using viral vectors to deliver transgenes in an uncontrolled fashion, we are working towards developing CRISPR genome editing to correct the RAGs disease-causing mutations by precisely modifying the genome. Here we compare the editing efficiency of RAG1 and RAG2 gRNAs in human HSPCs. Our results show that the chemically-modified sgRNAs and bipartite crRNA + tracrRNA systems, delivered as a ribonucleoprotein (RNP), enhance CRISPR genome editing in human HSPCs. Additionally, we show that we can use the combination of CRISPR-Cas9 RNP, chemically modified gRNAs, and recombinant adeno-associated viral vector (rAAV) donor transduction to effectively target functional RAG2 cDNA into the endogenous locus in human primary HSPCs. We will also present a comprehensive summary of the off-target effects associated with the delivery of the synthetic RAG1 and RAG2 gRNA forms. The off-target profiles for each class of gRNA will be compared using unbiased and highly sensitive method for detecting off-target events followed by quantification using a multiplexed-based, targeted sequencing approach. Finally, the implication of our findings for therapeutic genome editing will be discussed.