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. Author manuscript; available in PMC: 2020 Aug 21.
Published in final edited form as: Neuron. 2019 Jul 1;103(4):583–597.e8. doi: 10.1016/j.neuron.2019.05.047

Figure 1. Illustration of the HiUGE system.

Figure 1.

(A) Schematic of HiUGE method. The HiUGE donor vector expresses a donor-specific gRNA (DS-gRNA), which specifically recognizes the donor recognition sequence (DRS) and directs the cleavage and release of the donor payload for insertion into gene-specific gRNA (GS-gRNA) targeted loci for modification of proteins. (B) HiUGE donor vectors harboring short epitope tags employ a dual-orientation design for efficient expression following either forward or reverse genomic integration. A cassette of stop codons for all six ORFs (denoted by an “X” boxed in red, sequence listed in Table S1) is used to terminate translation. (C) Workflow using a small-scale, low-cost, and higher-throughput AAV supernatant production method for in vitro applications. (D-F) Proof-of-principle data showing HA-epitope KI to mouse Tubb3 gene in primary neuron culture of Cas9 mice after transduction with a combination of small-scale GS-gRNA AAV and HA-epitope donor AAV. (D) Representative (i) confocal and (ii) stimulated emission depletion (STED) images of immunocytochemistry staining for HA-epitope, showing the characteristic microtubule expression pattern of the HA-epitope labeled ßIII-tubulin. GFP fluorescence of the Cas9-2A-GFP, nuclei labeling with DAPI (4',6-diamidino-2-phenylindole), and synaptic marker Synapsin I staining are also shown. (E) Western blot for HA-epitope in a comparison against a negative control (transduced with empty GS-gRNA backbone and HiUGE donor), showing a single band of labeled protein at the expected molecular mass (~51kD) for ßIII-tubulin. (F) Representative sequencing results showing correct integration in forward and reverse orientation. Regions of DNA sequence containing genomic Tubb3, DRS (blue shade), restriction enzymes (yellow shade), HA-epitope (green shade) and stop codon cassette (red box) are indicated. Truncated terminal amino acids are noted. Translated amino acid sequences encoded by the payload and the in-frame stop codon (red asterisk) are indicated as well.