A multifunctional AAV-CRISPR-Cas9 and its host response.
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.
Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.|Biological and Biomedical Sciences Program, Harvard Medical School, Boston, Massachusetts, USA.
Wei Leong
0000-0002-4774-7959
WL
Chew
Biological and Biomedical Sciences Program, Harvard Medical School, Boston, Massachusetts, USA.|Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, USA.
Mohammadsharif
M
Tabebordbar
Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, USA.
Jason K W
JK
Cheng
Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
Prashant
P
Mali
Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, USA.
Elizabeth Y
EY
Wu
Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.|Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.
Alex H M
AH
Ng
Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, USA.|Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.
Kexian
K
Zhu
Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, USA.
Amy J
AJ
Wagers
Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.|Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.
George M
GM
Church
D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D000072669:Gene Editing; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D020133:Reverse Transcriptase Polymerase Chain Reaction