AAV-CRISPR gene editing is negated by pre-existing immunity to Cas9

Adeno-associated viral (AAV) vectors are a leading candidate

for the delivery of CRISPR-Cas9 for therapeutic genome editing

in vivo. However, AAV-based delivery involves persistent

expression of the Cas9 nuclease, a bacterial protein. Recent

studies indicate a high prevalence of neutralizing antibodies

and T cells specific to the commonly used Cas9 orthologs

from Streptococcus pyogenes (SpCas9) and Staphylococcus

aureus (SaCas9) in humans. We tested in a mouse model

whether pre-existing immunity to SaCas9 would pose a barrier

to liver genome editing with AAV packaging CRISPR-Cas9.

Although efficient genome editing occurred in mouse liver

with pre-existing SaCas9 immunity, this was accompanied by

an increased proportion of CD8+ T cells in the liver. This cytotoxic

T cell response was characterized by hepatocyte apoptosis,

loss of recombinant AAV genomes, and complete elimination

of genome-edited cells, and was followed by compensatory liver

regeneration. Our results raise important efficacy and safety

concerns for CRISPR-Cas9-based in vivo genome editing in

the liver.