AAV-CRISPR Gene Editing Is Negated by Pre-existing Immunity to Cas9

Mol Ther. 2020 Jun 3;28(6):1432-1441. doi: 10.1016/j.ymthe.2020.04.017. Epub 2020 Apr 19.


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.

Keywords: AAV-CRISPR; CD8+ T cell; SaCas9; adeno-associated virus; gene therapy; hepatocytes; immune response; liver; pre-existing immunity; somatic genome editing.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Biomarkers
  • CRISPR-Associated Protein 9 / adverse effects
  • CRISPR-Associated Protein 9 / immunology*
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Dependovirus / genetics*
  • Gene Editing / methods*
  • Gene Expression
  • Gene Order
  • Genetic Vectors / genetics*
  • Hepatocytes / metabolism
  • Humans
  • Immunization
  • Immunologic Memory
  • Immunophenotyping
  • Mice
  • RNA, Guide, CRISPR-Cas Systems
  • T-Lymphocyte Subsets / immunology
  • T-Lymphocyte Subsets / metabolism
  • Transgenes


  • Biomarkers
  • RNA, Guide, CRISPR-Cas Systems
  • CRISPR-Associated Protein 9