Postnatal Genome Editing With CRISPR

Abstract

Targeted genome editing holds tremendous promise for permanent correction of many genetic diseases. The recently developed CRISPR/Cas9 genome-editing tool exhibits facile programmability and robust gene-editing efficiency, and has been applied in cell cultures and animal tissues. However, multi-organ gene-editing in live mammals has not been examined or achieved. This study demonstrates genetic modification in multiple organs of postnatal mice by systemic delivery of CRISPR with adeno-associated viruses (AAVs). I resolved the AAV payload limitation by splitting Cas9 and reconstituting the native protein in vivo using scarless split-intein protein trans-splicing, which preserves full activity of Cas9. I determined that the delivery efficiency of AAV-CRISPR dictates gene-targeting rates in vivo, with the preferential gene-editing in liver and heart, and more modest editing efficiencies in skeletal muscle, brain and gonads, directly reflecting the infection profile of the virus serotype. To track CRISPR biodistribution, I established two reporter systems that apply in situ fluorescence activation to demarcate CRISPR-targeting events at single-cell resolution, identifying rare gene-edited cells that normally evade detection by sequencing. This exquisite detection sensitivity further allows evaluation of inter-generational transmission of gene-editing viruses. Finally, although Cas9 elicits host immune responses, these can be ameliorated by immunosuppression. I also identified a public Cas9-responsive T-cell clonotype and mapped the B-cell epitopes on Cas9 and AAV. Engineering tolerance to immunodominant epitopes may provide an avenue for avoiding immune rejection of AAV-CRISPR. The ability to create programmable genetic modifications in multiple organs of postnatal mammals provides a powerful tool for biological research, and foretells that the genomes of whole mammals may be rewritten at will.