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Nat Methods. 2016 Oct;13(10):868-74. doi: 10.1038/nmeth.3993. Epub 2016 Sep 5.

A multifunctional AAV-CRISPR-Cas9 and its host response.

Author information

1
Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
2
Biological and Biomedical Sciences Program, Harvard Medical School, Boston, Massachusetts, USA.
3
Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, USA.
4
Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.
5
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.
6
Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.

Abstract

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.

PMID:
27595405
PMCID:
PMC5374744
DOI:
10.1038/nmeth.3993
[Indexed for MEDLINE]
Free PMC Article

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