Plug-and-Play Protein Modification Using Homology-Independent Universal Genome Engineering.

Published

Journal Article

Analysis of endogenous protein localization, function, and dynamics is fundamental to the study of all cells, including the diversity of cell types in the brain. However, current approaches are often low throughput and resource intensive. Here, we describe a CRISPR-Cas9-based homology-independent universal genome engineering (HiUGE) method for endogenous protein manipulation that is straightforward, scalable, and highly flexible in terms of genomic target and application. HiUGE employs adeno-associated virus (AAV) vectors of autonomous insertional sequences (payloads) encoding diverse functional modifications that can integrate into virtually any genomic target loci specified by easily assembled gene-specific guide-RNA (GS-gRNA) vectors. We demonstrate that universal HiUGE donors enable rapid alterations of proteins in vitro or in vivo for protein labeling and dynamic visualization, neural-circuit-specific protein modification, subcellular rerouting and sequestration, and truncation-based structure-function analysis. Thus, the "plug-and-play" nature of HiUGE enables high-throughput and modular analysis of mechanisms driving protein functions in cellular neurobiology.

Full Text

Duke Authors

Cited Authors

  • Gao, Y; Hisey, E; Bradshaw, TWA; Erata, E; Brown, WE; Courtland, JL; Uezu, A; Xiang, Y; Diao, Y; Soderling, SH

Published Date

  • August 21, 2019

Published In

Volume / Issue

  • 103 / 4

Start / End Page

  • 583 - 597.e8

PubMed ID

  • 31272828

Pubmed Central ID

  • 31272828

Electronic International Standard Serial Number (EISSN)

  • 1097-4199

Digital Object Identifier (DOI)

  • 10.1016/j.neuron.2019.05.047

Language

  • eng

Conference Location

  • United States