Immunogenicity

Biology

Gene

Genetic enhancement

Genome editing

Cas9

Genome

Subgenomic mRNA

Computational biology

CRISPR

Genetics

Ribonucleoprotein

Immune system

Nature Methods

Packaging split Cas9 into AAVs increases cargo capacity and allows for efficient genome editing and gene activation in vivo. AAV–split-Cas9 activates the host immune system but does not trigger the extensive cellular damage observed with delivery of Cas9 via DNA electroporation. CRISPR–Cas9 delivery by adeno-associated virus (AAV) holds promise for gene therapy but faces critical barriers on account of its potential immunogenicity and limited payload capacity. Here, we demonstrate genome engineering in postnatal mice using AAV–split-Cas9, a multifunctional platform customizable for genome editing, transcriptional regulation, and other previously impracticable applications of AAV–CRISPR–Cas9. We identify crucial parameters that impact efficacy and clinical translation of our platform, including viral biodistribution, editing efficiencies in various organs, antigenicity, immunological reactions, and physiological outcomes. These results reveal that AAV–CRISPR–Cas9 evokes host responses with distinct cellular and molecular signatures, but unlike alternative delivery methods, does not induce extensive cellular damage in vivo. Our study provides a foundation for developing effective genome therapeutics.

A multifunctional AAV–CRISPR–Cas9 and its host response