In situ gene editing of hematopoietic stem cells via AAV-delivered CRISPR guide RNAs Open Access

• AAV8-mediated delivery of CRISPR gRNAs enables in situ modification of reporter alleles and disease-relevant loci in HSPCs.

• In situ HSPC modification efficiency varies with age, vector configuration, and guide copy number.

Hematopoietic stem cells (HSCs) are self-renewing, multipotent, and engraftable precursors of all blood cells. Efficient delivery of therapeutic gene products and gene editing machinery to correct disease-causing gene variants in endogenous HSCs while they remain in the body holds exciting potential to leverage HSC potency for the treatment of monogenic blood disorders. Towards this goal, we used adeno-associated virus (AAV) to deliver CRISPR guide RNAs (gRNAs) to edit HSC genomes in situ in Ai9;SpCas9-EGFP transgenics carrying a Cas9-activatable Lox-STOP-Lox (LSL)-tdTomato reporter cassette together with a constitutive SpCas9-2A-EGFP. Using a variety of conditions and vector designs, we tested whether systemic administration to these mice of AAVs carrying SpCas9 compatible gRNAs designed to cut DNA upstream and downstream of the STOP cassette would induce tdTomato expression in HSCs. Our findings identify self-complementary AAVs (scAAVs) and increased guide to Cas9 ratio as parameters facilitating higher editing efficiency. Of note, we find preserved multilineage output and engraftability of HSCs upon scAAV-gRNA editing. In an example application of this technology, we explore the potential for in situ HSC gene editing by dual AAV-CRISPR delivery and demonstrate robust gene modification, concurrent with induction of therapeutic fetal hemoglobin (HbF), in a sickle cell disease (SCD) mouse model modified to express SpCas9. In summary, this work offers a sensitive and adaptable platform that allows robust modification of HSC genomes in situ.